Biocompatible poly-beta-1-4-N-acetylglucosamine

ABSTRACT

The present invention relates to a purified, easily produced poly-β-1→4-N-acetylglucosamine (p-GlcNAc) polysaccharide species. The p-GlcNAc of the invention is a polymer of high molecular weight whose constituent monosaccharide sugars are attached in a β-1→4 conformation, and which is free of proteins, and substantially free of single amino acids, and other organic and inorganic contaminants. In addition, derivatives and reformulations of p-GlcNAc are described. The present invention further relates to methods for the purification of the p-GlcNAc of the invention from microalgae, preferably diatom, starting sources. Still further, the invention relates to methods for the derivatization and reformulation of the p-GlcNAc. Additionally, the present invention relates to the uses of pure p-GlcNAc, its derivatives, and/or its reformulations.

1. INTRODUCTION

The present invention relates, first, to a purified, easily producedpoly-β=1→4-N-acetylglucosamine (p-GlcNAc) polysaccharide species. Thep-GlcNAc of the invention is a polymer of high molecular weight whoseconstituent monosaccharide sugars are attached in a β-1→4 conformation,and which is free of proteins, and substantially free of single aminoacids, and other organic and inorganic contaminants. In addition,derivatives and reformulations of p-GlcNAc are described. The presentinvention further relates to methods for the purification of thep-GlcNAc of the invention from microalgae, preferably diatom, startingsources. Still further, the invention relates to methods for thederivatization and reformulation of the p-GlcNAc. Additionally, thepresent invention relates to the uses of pure p-GlcNAc, its derivatives,and/or its reformulations.

2. BACKGROUND OF THE INVENTION

There exists today an extensive literature on the properties,activities, and uses of polysaccharides that consist, in part, ofp-GlcNAc. A class of such materials has been generically referred to as“chitin”, while deacetylated chitin derivatives have been referred to as“chitosan”. When these terms were first used, around 1823, it wasbelieved that chitin and chitosan always occurred in nature as distinct,well-defined, unique; and invariant chemical species, with chitin beingfully acetylated and chitosan being fully deacetylated compositions. Itwas approximately a century later, however, before it was discoveredthat the terms “chitin” and “chitosan” are, in fact, very ambiguous.Rather than referring to well-defined compounds, these terms actuallyrefer to a family of compounds that exhibit widely differing physicaland chemical properties. These differences are due to the productsvarying molecular weights, varying degrees of acetylation, and thepresence of contaminants such as covalently bound, species-specificproteins, single amino acid and inorganic contaminants. Even today, theterms “chitin” and “chitosan” are used ambiguously, and actually referto poorly defined mixtures of many different compounds.

For example, the properties of “chitins” isolated from conventionalsources such as crustacean outer shells and fungal mycelial mats areunpredictably variable. Such variations are due not only to speciesdifferences but are also due to varying environmental and seasonaleffects that determine some of the biochemical characteristics of the“chitin”-producing species. In fact, the unpredictable variability ofraw material is largely responsible for the slow growth of chitin-basedindustries.

No reports exist today in the scientific literature describing theisolation and production, from material sources, of pure, fullyacetylated p-GlcNAc, i.e., a product or products uncontaminated byorganic or inorganic impurities. While McLachlan et al. (McLachlan, A.G. et al., 1965, Can. J. Botany 43:707-713) reported the isolation ofchitin, subsequent studies have shown that the “pure” substanceobtained, in fact contained proteins and other contaminants.

Deacetylated and partially deacetylated chitin preparations exhibitpotentially beneficial chemical properties, such as high reactivity,dense cationic charges, powerful metal chelating capacity, the abilityto covalently attach proteins, and solubility in many aqueous solvents.The unpredictable variability of these preparations, as described above,however, severely limits the utility of these heterogenous compounds.For example, the currently available “chitins” and “chitosans” give riseto irreproducible data and to unacceptably wide variations inexperimental results. Additionally, the available preparations are notsufficiently homogenous or pure, and the preparation constituents arenot sufficiently reproducible for these preparations to be acceptablefor use in applications, especially in medical ones. Thus, althoughextremely desirable, true, purified preparations of chitin and chitosan,whose properties are highly reproducible and which are easilymanufactured, do not currently exist.

3. SUMMARY OF THE INVENTION

The present invention relates, first, to an isolated, easily produced,pure p-GlcNAc species. The p-GlcNAc of the invention is a polymer ofhigh molecular weight whose constituent monosaccharides are attached ina β-1→4 conformation, and which is free of proteins, substantially freeof other organic contaminants, and substantially free of inorganiccontaminants.

The importance of the present invention resides in the fact that theproblem of unpredictable raw material variability has been overcome. Itis, for the first time, possible to produce, by simple means, and on acommercial scale, biomedically pure, p-GlcNAc of high molecular weightand consistent properties. The material produced in the presentinvention is highly crystalline and is produced from carefullycontrolled, aseptic cultures of one of a number of marine microalgae,preferably diatoms, which have been grown in a defined medium.

The present invention further describes derivatives and reformulationsof p-GlcNAc as well as methods for the production of such derivativesand reformulations. Such derivatizations may include, but are notlimited to polyglucosamine and its derivatives, and such reformulationsmay include, but are not limited to membranes, filaments, non-woventextiles, sponges, gels and three-dimensional matrices. Still further,the present invention relates to methods for the purification of thep-GlcNAc of the invention from microalgae, preferably diatom, sources.

Additionally, the present invention relates to the uses of the purifiedp-GlcNAc, its derivatives, and/or its reformulations. Among these usesare novel commercial applications relating to such industries as thebiomedical, pharmaceutical, cosmetic and agricultural industries, all ofwhich require starting materials of the highest degree of purity. Forexample, the p-GlcNAc materials of the invention may be formulated toexhibit controllable biodegradation properties, and, further, may beused as part of slow drug delivery systems, as cell encapsulationsystems, and as treatments for the prevention of post-surgicaladhesions; and for the induction of hemostasis. For example, thep-GlcNAc materials of the invention exhibit properties that make themideally suited for a large number of biomedical applications. Some ofthese properties include but are not limited to: high purity andcomposition consistency; biocompatibility; controllablebiodegradability; and, an ability to immobilize and encapsulate agents,such as therapeutic agents, and cells. These properties are useful, forexample, in the formulation of biodegradable barrier devices, improveddrug formulations and cell based therapeutics.

The biodegradable barriers of the invention include p-GlcNAc basedmaterials used as devices, for example, as temporary barriers whichbecome resorbed by the body. This category of products include, but isnot limited to, devices that prevent the formation of surgicaladhesions, stop bleeding, and promote wound healing. Such biodegradablebarriers can further be used as surgical space fillers, peridontalbarriers or for soft tissue augmentation.

Improved drug formulations of the invention include p-GlcNAc basedmaterials designed to deliver drugs. These new drug formulations are animprovement over traditional drug formulations, in that the drugformulations of the invention provide, for example, increasedeffectiveness, reduced toxicity and improved bioavailability. Theseimproved drug formulations can be used in combination with manytherapeutic agents including, but not limited to, chemotherapeuticagents, such as antitumor agents, as well as antibiotics,antibacterials, antifungals and anti-inflammatory drugs.

Additionally, the present invention relates to cell based therapeuticsusing p-GlcNAc based materials as a matrix for the encapsulation ofcells. For example, p-GlcNAc/cell encapsulations may be used for theimplantation of insulin-producing cells in the treatment of diabetes ordopamine-producing cells for the treatment of Parkinson's disease. Thep-GlcNAc cell encapsulations of the invention can also be used for thedelivery of cells to regenerate tissue such as, for example, skin,cartilage and bone.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Chemical structure of 100% p-GlcNAc. “n” refers to an integerranging from about 4,000 to about 150,000, with about 4,000 to about15,000 being preferred.

FIG. 2. Carbohydrate analysis of p-GlcNAc, Gas Chromatography-MassSpectroscopy data. Solid squares represent p-GlcNAc purified using theacid treatment/neutralization variation of the Chemical/Biologicalmethod, as described in Section 5.3.2, below.

FIG. 3A. Circular dichroism spectra of solid membranes of pure p-GlcNAc.

FIG. 3B. Circular dichroism spectra of solid membranes of Deacetylatedp-GlcNAc. The disappearance of the 211 nm minimum and 195 nm maximumobserved in pure p-GlcNAc (FIG. 3A) indicates complete deacetylationunder the conditions used, as described in Section 5.4 below.

FIG. 4A. Infra-red spectra analyses of thin membranes of pure diatomp-GlcNAc prepared by the mechanical force purification method, top, andthe chemical/biological purification method, bottom.

FIG. 4B. Infra-red spectra analyses of two preparations of commercial“chitin” cast into membranes according to the methods detailed inSection 5.5, below.

FIG. 4C. Infra-red spectra analyses of pure p-GlcNAc which was modifiedby heat denaturation (top) and by chemical deacetylation (bottom),according to the methods detailed in Section 5.4, below.

FIG. 4D. Infra-red spectrum analysis of a p-GlcNAc membrane derived fromthe diatom Thalassiosira fluviatilis, using the chemical/biologicalpurification method, as detailed in Section 5.3.2, below.

FIG. 4E. Infra-red spectrum analysis of a p-GlcNAc membrane prepared bythe mechanical force purification method, as described in Section 5.3.1,below, following autoclaving.

FIG. 5A. NMR analysis of p-GlcNAc purified using the chemical/biologicalpurification method as described in Section 5.3.2, below. Chartdepicting peak amplitudes, areas, and ratios relative to referencecontrols. Ratio of total areas of peaks.

FIG. 5B. NMR analysis of p-GlcNAc purified using the chemical/biologicalpurification method as described in Section 5.3.2. The graph depicts theratios of total areas of peaks.

FIG. 6. Transmission electron micrographs (TEM) of a p-GlcNAc membraneprepared by the mechanical force purification method as described inSection 5.3.1, below. Magnification: top, 4190×; bottom, 16,250×.

FIG. 7. Transmission electron micrographs (TEM) of a p-GlcNAc membraneby HF treatment as described in the discussion of thechemical/biological purification method in Section 5.3.2, below.Magnification: top, 5270×; bottom, 8150×.

FIG. 8. Transmission electron micrographs (TEM) of a p-GlcNAc membraneprepared by the acid treatment/neutralization variation of thechemical/biological purification method, as described in Section 5.3.2,below. Magnification: top, 5270×; bottom, 16,700×.

FIG. 9A. Scanning electron micrograph depicting a p-GlcNAc membraneprepared by the acid treatment/neutralization variation of thechemical/biological purification method as described in Section 5.3.2,below. Magnification: 200×.

FIG. 9B. Scanning electron micrograph depicting a p-GlcNAc membraneprepared by the acid treatment/neutralization variation of thechemical/biological purification method as described in Section 5.3.2,below. Magnification: 1000×.

FIG. 9C. Scanning electron micrograph depicting a p-GlcNAc membraneprepared by the acid treatment/neutralization variation of thechemical/biological purification method as described in Section 5.3.2,below. Magnification: 5000×.

FIG. 9D. Scanning electron micrograph depicting a p-GlcNAc membraneprepared by the acid treatment/neutralization variation of thechemical/biological purification method as described in Section 5.3.2,below. Magnification: 10,000×.

FIG. 9E. Scanning electron micrograph depicting a p-GlcNAc membraneprepared by the acid treatment/neutralization variation of thechemical/biological purification method as described in Section 5.3.2,below. Magnification: 20,000×.

FIG. 10. Scanning electron micrographs of a pure p-GlcNAc. membrane madefrom material which was initially produced using the celldissolution/neutralization purification method described in Section 5.3,below, dissolved in dimethylacetamide/lithium chloride, andreprecipitated in H₂O into a mat, as described below in Section 5.5.Magnification: top, 1000×, bottom, 10,000×.

FIG. 11. Scanning electron micrographs of a deacetylated p-GlcNAc mat.Magnification: top, 1000×, bottom, 10,000×.

FIG. 12. Photographs of diatoms. Note the p-GlcNAc fibers extending fromthe diatom cell bodies.

FIG. 13. Diagram depicting some of the possible p-GlcNAc anddeacetylated p-GlcNAc derivatives of the invention. (Adapted from S.Hirano, “Production and Application of Chitin and Chitosan in Japan”, in“Chitin and Chitosan”, 1989, Skjak-Braek, Anthonsen, and Sanford, eds.Elsevier Science Publishing Co., pp. 37-43.)

FIG. 14. Cell viability study of cells grown in the presence or absenceof p-GlcNAc membranes. Closed circle (●): cells grown on p-GlcNAcmatrix; open circles (◯): cells grown in absence of matrix.

FIG. 15. SEM micrographs of transformed mouse fibroblast cells grown onp-GlcNAc membranes. Magnification: top, 100×; bottom, 3000×.

FIG. 16A. Scanning electron micrograph (SEM) of a collagen-only controlmaterial prepared according to the method described, below, in Section13.1. Magnification 100×.

FIG. 16B. Scanning electron micrograph (SEM) of a collagen/p-GlcNAchybrid material prepared according to the method described, below, inSection 13.1. Ratio collagen suspension:p-GlcNAc suspension equals 3:1,with final concentrations of 7.5 mg/ml collagen and 0.07 mg/ml p-GlcNAc.Magnification 100×.

FIG. 16C. Scanning electron micrograph (SEM) of a collagen/p-GlcNAchybrid material prepared according to the method described, below, inSection 13.1. Ratio collagen suspension:p-GlcNAc suspension equals 1:1,with final concentrations of 5.0 mg/ml collagen and 0.12 mg/ml p-GlcNAc.Magnification 100×.

FIG. 16D. Scanning electron-micrograph (SEM) of a collagen/p-GlcNAchybrid material prepared according to the method described, below, inSection 13.1. Ratio collagen suspension:p-GlcNAc suspension equals 2:2,with final concentrations of 10.0 mg/ml collagen and 0.25 mg/mlp-GlcNAc. Magnification 100×.

FIG. 16E. Scanning electron micrograph (SEM) of a collagen/p-GlcNAchybrid material prepared according to the method described, below, inSection 13.1. Ratio collagen suspension:p-GlcNAc suspension equals 1:3,with final concentrations of 2.5 mg/ml collagen and 0.25 mg/ml p-GlcNAc.Magnification 100×.

FIG. 17A. SEM of mouse 3T3 fibroblast cells cultured on thecollagen-only control material of FIG. 16A, above. Magnification 100×.

FIG. 17B. SEM of mouse 3T3 fibroblast cells cultured on thecollagen/p-GlcNAc material of FIG. 16B, above. Magnification 100×.

FIG. 17C. SEM of mouse 3T3 fibroblast cells cultured on thecollagen/p-GlcNAc material of FIG. 16C, above. Magnification 100×.

FIG. 17D. SEM of mouse 3T3 fibroblast cells cultured on thecollagen/p-GlcNAc material of FIG. 16D, above. Magnification 100×.

FIG. 18. Transformed NMR data curves, used to obtain areas for eachcarbon atom and to then calculate the CH3 (area) to C-atom (area)ratios.

FIG. 19. Typical p-GlcNAc C¹³-NMR spectrum. The individual peaksrepresent the contribution to the spectrum of each unique carbon atom inthe molecule.

FIG. 20. Transformed NMR spectrum data representing values calculatedfor CH3 (area) to C-atom (area) ratios. Top: Graphic depiction of data;bottom: numerical depiction of data.

FIG. 21A-G. Three-dimensional p-GlcNAc matrices produced in varioussolvents. Specifically, the p-GlcNAc matrices were produced in distilledwater (FIG. 21A, FIG. 21D), 10% methanol in distilled water (FIG. 21B),25% methanol in distilled water (FIG. 21C), 10% ethanol in distilledwater (FIG. 21E), 25% ethanol in distilled water (FIG. 21F) and 40%ethanol in distilled water (FIG. 21G). Magnification: 200×. A scalemarking of 200 microns is indicated on each of these figures.

FIG. 22A-G. Fibroblast cells grown on three-dimensional p-GlcNAcmatrices prepared by lyophilizing p-GlcNAc in distilled water.Magnification: 100× (FIGS. 22A, 22E), 500× (FIG. 22B), 1000× (FIGS. 22C,22F), 5000× (FIGS. 22D, 22G). Scales marking 5, 20, 50, or 200 microns,as indicated, are included in each of the figures.

FIG. 23. A typical standard curve obtained using the proceduredescribed, below, in Section 18.1. A standard curve such as this one wasused in the lysozyme-chitinase assay also described, below, in Section18.1.

FIG. 24. p-GlcNAc lysozyme digestion data. The graph presented heredepicts the accumulation of N-acetylglucosamine over time, as p-GlcNAcmembranes are digested with lysozyme. The graph compares the degradationrate of fully acetylated p-GlcNAc to partially (50%) deacetylatedp-GlcNAc, and demonstrates that the degradation rate for the partiallydeacetylated p-GlcNAc was substantially higher than that of the fullyacetylated p-GlcNAc material.

FIG. 25. p-GlcNAc lysozyme digestion data. The graph presented heredepicts the accumulation of N-acetylglucosamine over time, as p-GlcNAcmembranes are digested with lysozyme. The graph compares the degradationrate of two partially deacetylated p-GlcNAc membranes (specifically a25% and a 50% deacetylated p-GlcNAc membrane). The data demonstrate thatthe degradation rate increases as the percent of deacetylationincreases, with the degradation rate for the 50% deacetylated p-GlcNAcmembrane being substantially higher than that of the 25% deacetylatedp-GlcNAc membrane.

FIG. 26A-26E. p-GlcNAc in vivo biodegradability data. FIG. 26A-26Cdepict rats which have had prototype 1 (fully acetylated p-GlcNAc)membrane abdominally implanted, as described, below, in Section 18.1.FIG. 26A shows a rat at day 0 of the implantation; FIG. 26B shows a ratat day 14 post-implantation; FIG. 26C shows a rat at day 21post-implantation. FIG. 26D-26E depict rats which have had prototype 3A(lyophilized and partially deacetylated p-GlcNAc membrane) abdominallyimplanted, as described, below, in Section 18.1. FIG. 26D shows a rat atday 0 of the implantation; FIG. 26E shows a rat at day 14post-implantation.

FIG. 27. The graph depicted here illustrates data concerning the percentincrease in tumor size of animals which either received no treatment (●)or received p-GlcNAc-lactate/fluorouracil (5′-FU) (◯), as described,below, in Section 20.1.

FIG. 28. The graph depicted here illustrates data concerning the percentincrease in tumor size of animals which either received p-GlcNAc-lactatealone (●) or received p-GlcNAc-lactate/fluorouracil (5′-FU) (◯), asdescribed, below, in Section 20.1.

FIG. 29. The graph depicted here illustrates data concerning the percentincrease in tumor size of animals which either received no treatment (●)or received p-GlcNAc-lactate/mitomycin (mito) (◯), as described, below,in Section 20.1.

FIG. 30. The graph depicted here illustrates data concerning the percentincrease in tumor size of animals which either received p-GlcNAc-lactatealone (●) or received p-GlcNAc-lactate/mitomycin (mito) (◯), asdescribed, below, in Section 20.1.

FIG. 31. The bar graph depicted here illustrates the average percentchange in tumor size per animal of animals treated with p-GlcNAc/5′-FUhigh dose (bar 1), p-GlcNAc/5′-FU low dose (bar 2), p-GlcNAc membranealone (bar 3), and untreated (bar 4). N=4 for bars 1 and 2, n=2 for bars3 and 4.

FIG. 32A. Photograph of p-GlcNAc-treated site of Yorkshire pig abdomen21 days after four 2.0×2.0 cm serosal abrasion lesions were created.

FIG. 32B. Photograph of non-treated-control site of Yorkshire pigabdomen 21 days after four 2.0×2.0 cm serosal abrasion lesions werecreated.

5. DETAILED DESCRIPTION OF THE INVENTION

Presented below, is, first, a description of physical characteristics ofthe purified p-GlcNAc species of the invention, of the p-GlcNAcderivatives, and of their reformulations. Next, methods are describedfor the purification of the p-GlcNAc species of the invention frommicroalgae, preferably diatom, starting sources. Third, derivatives andreformulations of the p-GlcNAc, and methods for the production of suchderivatives and reformulations are presented. Finally, uses arepresented for the p-GlcNAc, p-GlcNAc derivatives and/or p-GlcNAcreformulations of the invention.

5.1. p-GlcNAc

The p-GlcNAc polysaccharide species of the invention is a polymer ofhigh molecular weight ranging from a weight average of about 800,000daltons to about 30 million daltons, based upon gel permeationchromatography measurements. Such a molecular weight range represents ap-GlcNAc species having about 4,000 to about 150,000 N-acetylglucosaminemonosaccharides attached in a β-1→4 configuration, with about 4,000 toabout 15,000 N-acetylglucosamine monosaccharides being preferred (FIG.1).

The variability of the p-GlcNAc of the invention is very low, and itspurity is very high, both of which are evidenced by chemical andphysical criteria. Among these are chemical composition andnon-polysaccharide contaminants. First, chemical composition data forthe p-GlcNAc produced using two different purification methods, both ofwhich are described in Section 5.3, below, is shown in Table I below. Ascan be seen, the chemical composition of the p-GlcNAc produced by bothmethods is, within the bounds of experimental error, the same as theformula compositions of p-GlcNAc. Second, as is also shown in Table I,the p-GlcNAc produced is free of detectable protein contaminants, issubstantially free of other organic contaminants such as free aminoacids, and is substantially free of inorganic contaminants such as ashand metal ions (the p-GlcNAc of the invention may deviate up to about 2%from the theoretical values of carbon, hydrogen, nitrogen and oxygen forpure p-GlcNAc). Therefore, as used herein, the terms “substantially freeof organic contaminants” and “substantially free of inorganiccontaminants” refer to compositions of p-GlcNAc having the profiles forcarbon, hydrogen, nitrogen and oxygen which deviate no more than about2% from the theoretical values, and preferably, the p-GlcNAc of theinvention contain a profile as exemplified in the Experimental Data onp-GlcNAc mats in Table I (allowing for the percent deviation). Further,the p-GlcNAc of the invention exhibits a very low percentage of boundwater. TABLE I CHEMICAL ANALYSIS DATA (% by weight) Theoretical Valuesfor Pure p-GlcNAC: Carbon 47.29 Hydrogen 6.40 Nitrogen 6.89 Oxygen 39.41Protein 0.00

Experimental Data on p-GlcNAc Mats

(Number of Experimental Batches for each Membrane Type Being Greaterthan 30 for each Membrane Type) CHEMICAL/ MECHANICAL FORCE BIOLOGICALMETHOD METHOD Normalized¹ % Dev. Normalized¹ % Dev. Carbon 47.21 ± 0.08−0.17 47.31 ± 0.11 +0.04 Hydrogen  6.45 ± 0.08 +0.78  6.34 ± 0.08 −0.94Nitrogen  6.97 ± 0.18 +0.87  6.94 ± 0.16 +0.73 Oxygen 39.55 ± 0.36 +0.3639.41 ± 0.10 0.00 Average Values Average Values Protein 0.00 0.00 Ash1.30 0.98 Moisture 2.0 1.2¹Raw analytical data have been normalized to account for ash andmoisture content of the samples.

The pure p-GlcNAc of the invention exhibits a carbohydrate analysisprofile substantially similar to that shown in FIG. 2. The primarymonosaccharide of the pure p-GlcNAc of the invention isN-acetylglucosamine. Further, the pure p-GlcNAc of the invention doesnot contain the monosaccharide glucosamine.

The circular dichroism (CD) and sharp infra-red spectra (IR) of thep-GlcNAc of the invention are shown in FIG. 3A, and FIGS. 4A and 4D,respectively, which present analyses of material produced using themethods described in Section 5.3, below. Such physical data corroboratesthat the p-GlcNAc of the invention is of high purity and crystallinity.The methods used to obtain the CD and IR data are described, below, inthe Working Example in Section 6.

NMR analysis of the pure p-GlcNAc of the invention exhibits a patternsubstantially similar to that seen in FIGS. 5A, 5B, 18A and 18B. Such anNMR pattern indicates not only data which is consistent with thep-GlcNAc of the invention being a fully acetylated polymer, but alsodemonstrates the lack of contaminating organic matter within thep-GlcNAc species.

The electron micrographic structure of the p-GlcNAc of the invention, asproduced using the methods described in Section 5.3, below anddemonstrated in the Working Examples presented, below, in Section 8 and9, is depicted in FIG. 6 through FIG. 9E.

The p-GlcNAc of the invention exhibits a high degree ofbiocompatability. Biocompatability may be determined by a variety oftechniques, including, but not limited to such procedures as the elutiontest, intramuscular implantation, or intracutaneous or systemicinjection into animal subjects. Briefly, an elution test (U.S.Pharmacopeia XXII, 1990, pp. 1415-1497; U.S. Pharmacopeia XXII, 1991,Supplement 5, pp. 2702-2703) is designed to evaluate thebiocompatability of test article extracts, and assays the biologicalreactivity of a mammalian cell culture line which is sensitive toextractable cytotoxic articles (such as, for example, the L929 cellline) in response to the test article. The Working Example presented inSection 10, below, demonstrates the high biocompatability of thep-GlcNAc of the invention.

5.2. Methods of Producing Microalgal Sources of βp-GlcNAc 5.2.1.Microalgal Sources of p-GlcNAc

The p-GlcNAc of the invention is produced by, and may be purified from,microalgae, preferably diatoms. The diatoms of several genuses andnumerous species within such genuses may be utilized as p-GlcNAcstarting sources. Each of these diatoms produce p-GlcNAc. See FIG. 12for photographs of such diatoms. The diatoms which may be used asstarting sources for the production of the p-GlcNAc of the inventioninclude, but are not limited to members of the Coscinodiscus genus, theCyclotella genus, and the Thalassiosira genus, with the Thalassiosiragenus being preferred.

Among the Coscinodiscus genus, the species of diatom that may be used toproduce the p-GlcNAc of the invention include, but are not limited tothe concinnus and radiatus species. The diatoms among the Cyclotellagenus which may be used include, but are not limited to the caspia,cryptica, and meneghiniana species. The Thalassiosira diatoms that maybe utilized to produce the starting material for the p-GlcNAc of theinvention include, but are not limited to the nitzschoides, aestivalis,antarctica, deciphens, eccentrics, floridana, fluviatilis, gravida,guillardii, hyalina, minima, nordenskioldii, oceanica, polychorda,pseudonana; rotula, tubifera, tumida, and weissflogii species, with thefluviatilis and weissflogii species being preferred.

Diatoms such as those described above may be obtained, for example, fromthe culture collection of the Bigelow Laboratory for Ocean Sciences,Center for Collection of Marine Phytoplankton (McKown Point, WestBoothbay Harbor, Me., 04575).

5.2.2. Methods for Growing Diatoms

Any of the diatoms described in Section 5.2.1, above, may be grown byutilizing, for example, the methods described in this section. Newdiatom cultures are initiated by inoculating, under aseptic conditions,Nutrient Medium with an aliquot of a mature diatom culture. The NutrientMedium must be free of all other microorganisms, therefore allmaterials, including water, organic components, and inorganic componentsused in the preparation of the Nutrient Medium must be sterile. Inaddition, it is mandatory that all procedures involved in this operationbe conducted under strictly aseptic conditions, i.e., all containers,all transfers of substances from one vessel to another, etc. must beperformed in a sterile environment. The quantity of Nutrient Medium tobe prepared at one time should not exceed what is necessary to start anew culture. For example, Fernbach flasks which occupy approximately onesquare foot of surface may be used as vessels for the diatom cultures,and such vessels require one liter of Nutrient Medium for optimum growthof the diatom organism.

Preparation of the nutrient medium involves the following operations:

-   -   a) Acquisition and processing of seawater    -   b) Preparation of distilled and deionized water.    -   c) Preparation of primary nutrient stocks    -   d) Preparation of nutrient working stocks    -   e) Preparation of the final nutrient medium

Filtered seawater may be obtained, for example, from the Marine BiologyLaboratory (Woods Hole, Mass.). Seawater containers should be stored at5° C. (+ or −2° C.). When required, the necessary volume of water may befiltered through a Buchner filtration unit, using a Supor-800 polyethersulfone filter membrane with 0.8 micron pore size (Gelman, Inc.). Theseawater is then sterilized by autoclaving at, for example, 121° C. forat least about 15 minutes per liter. On completion of the sterilizationprocess, the capped flasks are immediately cooled, preferably bytransfer to a cold room capable of allowing the solutions to reach atemperature of approximately 5° C. (+ or −2° C.). When it is to be used,solutions are allowed to reach room temperature.

Tap water is distilled and deionized using standard equipment andprocedures, and collected and stored in clean, securely capped,preferably glass, containers.

Listed below are formulas which may be followed in preparing the stocksolutions necessary for the preparation of the Nutrient Medium. It is tobe understood that while such formulas are to be used as guides, it isintended that routine variations of such formulas which contribute tothe preparation of a Nutrient Medium capable of sustaining microalgaldiatom growth sufficient for the p-GlcNAc preparative processesdescribed here also be within the scope of the present invention.

I. Trace Metal Primary Stocks (TMPS)

-   -   a. 39 mM CuSO₄.5H₂O (copper [II] sulfate pentahydrate) (9.8 g        copper [II] sulfate/L)    -   b. 7.5 mM ZnSO₄.7H₂O (Zinc sulfate heptahydrate) (22 g zinc        sulfate/L)    -   c. 42 mM CoCl₂.6H₂O (Cobalt [II] chloride hexahydrate) (10 g        cobalt [II] chloride/L)    -   d. 91 mM MnCl₂.4H₂O (Manganese [II] chloride tetrahydrate) 18 g        manganese [II] chloride/L)    -   e. 26 mM NaMoO₄.2H₂O (Sodium molybdate dihydrate) 6.3 g sodium        molybdate/L)    -   f. 1 mM H₂SeO₃ (Selenious acid) (0.129 g selenious acid/L).        Sterile filter each nutrient with a filter of no greater than        0.2 mm pore size.

II. Vitamin Primary Stocks (VPS)

-   -   a. 1 mg/ml Vitamin B12    -   b. 0.1 mg/ml Biotin        Sterile filter both stocks with a filter of no greater than 0.2        mm pore size.

III. Sodium Salts Working Stocks (SSWS)

-   -   a. Sodium nitrate working stock: 0.88 M (75 g NaNO₃/L)    -   b. Sodium phosphate monobasic monohydrate working stock: 36.2 mM        NaH₂PO₄.H₂O (5 g NaH₂PO₄.H₂O/L)    -   c. Sodium metasilicate monohydrate working stock: 0.11 M        Na₂SiO₃.9H₂O (30 g Na₂SiO₃.9H₂O/L)        Sterile filter each of the SSWS with a filter of no greater than        0.2 mm pore size.

IV. Trace Metal Working Stocks (TMWS)

-   -   11.7 mM Na₂EDTA (Ethylenediamine Tetraacetic acid, disodium salt        dihydrate) (4.36 g/L)    -   11.7 mM FeCl₃.6H₂O (Iron [III] chloride hexahydrate) (3.15 g/L)    -   1 ml/L of each of the six primary trace metal stocks listed        above.        Sterile filter with a filter of no greater than 0.2 mm pore        size. Note that the trace metal working stock must be prepared        fresh weekly.

V. vitamin Working Stock (VWS)

-   -   1.0 μg/ml Biotin (1.0 ml primary Biotin Stock/100 ml)    -   1.0 μg/ml Vitamin B12 (0.1 ml Vitamin B12 primary stock/100 ml)    -   20 mg of Thiamine HCl (Thiamine hydrochloride/100 ml).        Sterile filter with a filter of no greater than 0.2 mm pore        size. Note that a new Vitamin Working Stock should be prepared        fresh weekly.

Described below are techniques which may be followed for the preparationof Nutrient Medium and for diatom culturing. It is to be understoodthat, in addition to these techniques, any routine variation in theformulas and/or procedures described herein which result in a NutrientMedium and in procedures capable of sustaining diatom growth sufficientfor the preparative processes described herein is intended to be withinthe scope of the present invention.

Nutrient Medium may be prepared, for example, as follows: To each literof filtered and sterilized seawater may be added 1 ml of the NaNO₃working stock, 1 ml of the NaH₂PO4.H₂O working stock, 1 ml of the TraceMetal working stock, and 1 ml of the Na₂SiO₃.9H₂O working stock.Simultaneously with the addition of Na₂SiO₃9H₂O, 2 mls of 1 N HCl may beadded and the solution may be shaken to mix. Next, 1.5 mls 1 N NaOH maybe added and the solution may again be shaken to mix. Finally, 0.5 ml ofthe Vitamin working stock may be added.

In order to grow a new diatom culture, 7 ml of a mature culture, (havinga cell density within a range of about 1×10⁵ to about 1×10⁶ cells/ml.),may be transferred to a sterile container containing 100 ml of sterileNutrient Medium, which may be prepared according to the methodsdescribed above. The inoculated culture may then be incubated for 8 daysunder the following conditions:

-   -   Temperature: 20 degrees Centigrade Constant illumination.    -   Agitation: Gentle swirling of flasks once per day.

After 8 days of incubation, 80 ml of this incubated culture may betransferred, under sterile conditions, to 1000 ml of Nutrient Medium,which may, for example, be contained in a 2.8 L Fernbach flask,protected by a cotton wool plug covered by cheesecloth. Such a culturemay be allowed to incubate and grow to the desired cell density, oralternatively, may be used to inoculate new diatom cultures. Once aculture reaches a desired cell density, the culture's p-GlcNAc fibersmay be harvested, and the p-GlcNAc of the invention may be purified,using methods such as those described below in Section 5.3, below.

CO₂ may be dissolved in the culture solution in order to maintain aculture pH of approximately 7 to 8, with approximately 7.4 beingpreferred. The maintenance of such a neutral pH environment, greatlyincreases the p-GlcNAc yield that may be obtained from each diatomculture.

5.3. Methods for Isolation, Purification, and Concentration of p-GlcNAcFibers

Presented in this Section are methods which may be utilized for thepreparation of p-GlcNAc fibers from diatom cultures such as thosedescribed, above, in Section 5.2.

While each of the methods described below for the purification ofp-GlcNAc from microalgae, preferably diatom, starting sources producesvery pure, unadulterated, crystalline p-GlcNAc, each of the methodsyields p-GlcNAc having specific characteristics and advantageousfeatures. For example, the p-GlcNAc of the invention purified via theMechanical Force method presented in Section 5.3.1, below, produces ap-GlcNAc membrane that provides a superior substrate for the attachmentof cells to the p-GlcNAc. The second method, described below in Section5.3.2, the Chemical/Biological method, produces a much higher averageyield than the average p-GlcNAc yield produced by the Mechanical Forcemethod. Additionally, the acid treatment/neutralization variationdescribed as part of the Chemical/Biological method of Section 5.3.2,below, produces extremely long p-GlcNAc fibers, with some fibers beingin excess of 100 μm, and containing molecules of the p-GlcNAc polymer ofvery high molecular weight, as high as 20-30 million daltons.

5.3.1. Mechanical Force Method for Preparation of Pure p-GlcNAc

The p-GlcNAc fibers may be separated from diatom cell bodies bysubjecting the contents of the culture to an appropriate mechanicalforce. Such a mechanical force may include, but is not limited to, ashear force generated by, for example, a colloid mill, an ultrasounddevice, or a bubble generator, or a cutting force generated by, forexample, a Waring blender.

The resulting suspension of diatom cell bodies and p-GlcNAc fibers arethen segregated. For example, the suspension may be subjected to aseries of centrifugation steps which segregate the p-GlcNAc fibers fromthe cell bodies, yielding a clear supernatant exhibiting little, if any,visible flocculent material. A fixed angle rotor, and a temperature ofabout 10° C. are preferred for the centrifugation steps. The speed,duration, and total number of centrifugation steps required may varydepending on, for example, the specific centrifugation rotor being used,but the determination of the values for such parameters will be apparentto one of ordinary skill in the art.

The p-GlcNAc fibers in the supernatant may then be concentrated usingtechniques well known to those of skill in the art. Such techniques mayinclude, but are not limited to suction and filtration devices.

Finally, the concentrated p-GlcNAc fibers are washed with, for example,distilled-deionized water, HCl and ethanol, or other appropriatesolvents, preferably solvents, such as alcohols, in which both organicand inorganic materials dissolve.

The Working Example presented in Section 7, below, demonstrates the useof this method for the purification of p-GlcNAc.

5.3.2. Chemical/Biological Method for Purification of p-GlcNAc

In this method, p-GlcNAc fibers are separated from diatom cell bodies bysubjecting them to chemical and/or biological agents as described inmore detail below.

Diatom cultures may be treated with a chemical capable of weakeningdiatom cell walls, which leads to a release of the p-GlcNAc fiberswithout altering their structure. Such a chemical may include, but isnot limited to, hydrofluoric acid (HF). Alternatively, a mature diatomculture may be treated with a biological agent capable of altering abiological process may be used to inhibit p-GlcNAc fiber synthesis, thusreleasing the fibers already present. For example, such an agent mayinclude, but is not limited to, polyoxin-D, an inhibitor of the enzymeN-acetylglucosaminyl-P-transferase.

The cell bodies and p-GlcNAc-containing fibers of diatom culturestreated with a member of the above described chemical or biologicalagents are then segregated. For example, the contents of treated diatomcultures may be allowed to settle such that the contents of the culturesare allowed to form two distinct layers. The upper layer will containprimarily the p-GlcNAc fibers, while the bottom layer will contain thecell bodies. The upper p-GlcNAc fiber-containing layer may be siphonedoff, leaving behind the settled cellular material of the bottom layer.

The siphoned off p-GlcNAc fiber-containing layer may then be furtherpurified to remove protein and other unwanted matter by treatment with adetergent that will not damage the p-GlcNAc fibers. Such a detergent mayinclude, but is not limited to, sodium dodecyl sulfate (SDS).

When acid treatment, such as HF treatment, is used to separate p-GlcNAcfibers from diatom cell bodies, a step may be included for the dispersalof the fibers. Such a step may include, but is not limited to, the useof mechanical force for fiber dispersal, such as a step in which thefibers are subjected to the movements of an orbital shaker.

Alternatively, the acid-treated suspension may, in an optional step, beneutralized prior to further purification by detergent treatment. Suchneutralization will, in general, change the pH of the suspension fromapproximately 1.8 to approximately 7.0, and may be accomplished by, forexample, the addition of an appropriate volume of 1M Tris (pH 8.0) orthe addition of an appropriate volume of sodium hydroxide (NaOH).Neutralization, in general, yields pure p-GlcNAc fibers of asubstantially greater length than the other purification methodsdiscussed herein.

The purified p-GlcNAc fibers may then be concentrated using techniqueswell known to those of skill in the art, such as by utilizing a suctionand filtration device. Finally, the p-GlcNAc fibers are washed, in aseries of steps with distilled-deionized water, HCl and ethanol, orother appropriate solvents, preferably solvents, such as alcohols, inwhich both organic and inorganic materials dissolve.

The Working Example presented, below, in Section 8 demonstrates thesuccessful utilization of such a purification method.

5.4. Derivatization of p-GlcNAc

The pure, fully acetylated p-GlcNAc of the invention may be derivatized,by utilizing a variety of controlled conditions and procedures, into alarge range of different compounds. See FIG. 13 for a diagram depictingsome of these compounds. Such derivatized compounds may include, but arenot limited to, partially or completely deacetylated p-GlcNAc, which hasbeen modified via chemical and/or enzymatic means, as described infurther detail, below. Additionally, p-GlcNAc, or its deacetylatedderivative, may be derivatized by being sulfated, phosphorylated, and/ornitrated. Further, as detailed below, O-sulfonyl, N-acyl, O-alkyl,N-alkyl, deoxyhalogen, and N-alkylidene and N-arylidene and otherderivatives may be prepared from the p-GlcNAc or deacetylated p-GlcNAcof the invention. The deacetylated p-GlcNAc of the invention may also beused to prepare a variety of organic salts and/or metal chelates.Further, the p-GlcNAc, or a derivative thereof, of the invention mayhave attached to it, either covalently or non-covalently, any of avariety of molecules. Still further, the p-GlcNAc of the invention, or aderivative thereof, may be subjected to controlled hydrolysis conditionswhich yield groups of molecules having uniform and discrete molecularweight characteristics.

One or more of the monosaccharide units of the p-GlcNAc of the inventionmay be deacetylated to form a poly-β-1→4-N-glucosamine species. Apoly-β-1→4-N-glucosamine species of the invention in which each of themonosaccharide units of the poly-β-1→4-N-acetylglucosamine species ofthe invention has been deacetylated will have a molecular weight ofabout 640,000 daltons to about 24 million daltons, with about 640,000daltons to about 2.4 million daltons being preferred. A species withsuch a molecular weight range represents a species having about 4000 toabout 150,000 glucosamine monosaccharides covalently attached in a β-1→4configuration, with about 4,000 to about 15,000 glucosaminemonosaccharides being preferred. At least one of the monosaccharideunits of the poly-β-1→4-N-glucosamine species may remain acetylated,with about 25% to about 75% acetylation being preferred, and about 30%acetylation being most preferred.

The p-GlcNAc of the invention may be deacetylated by treatment with abase to yield glucosamines with free amino groups. This hydrolysisprocess may be carried out with solutions of concentrated sodiumhydroxide or potassium hydroxide at elevated temperatures. To preciselycontrol the extent of deacetylation and to avoid degradation of the maincarbohydrate chain of the polysaccharide molecule, however, it ispreferable that an enzymatic procedure utilizing a chitin deacetylaseenzyme be used for p-GlcNAc deacylation. Such a deacetylase enzymaticprocedure is well known to those of skill in the art and may beperformed as in (U.S. Pat. No. 5,219,749), which is incorporated herein,by reference, in its entirety.

One or more of the monosaccharide units of the p-GlcNAc of the inventionmay be derivatized to contain at least one sulfate group, or,alternatively, may be phosphorylated or nitrated, as depicted below:

where, R and/or R₁, in place of a hydrogen, and/or R₂, in place of—COCH₃, may be a sulfate (—SHO₃), a phosphate (—P(OH)₂), or a nitrate(—NO₂) group.

Described below are methods by which such p-GlcNAc derivatives may beprepared. Before performing methods such as those described in thisSection, it may be advantageous to first lyophilize, freeze in liquidnitrogen, and pulverize the p-GlcNAc starting material.

Sulphated p-GlcNAc derivatives may be generated, by, for example, a twostep process. In the first step, O-carboxymethyl p-GlcNAc may beprepared from the p-GlcNAc and/or p-GlcNAc derivatives of the inventionby, for example, utilizing techniques such as those described by Tokuraet al. (Tokura, S. et al., 1983, Polym. J. 15:485). Second, thesulfation step may be carried out with, for example,N,N-dimethyl-formamide-sulfur trioxide, according to techniques wellknown to those of skill in the art, such as are described by Schweiger(Schweiger, R. G., 1972, Carbohydrate Res. 21:219). The resultingproduct may be isolated as a sodium salt.

Phosphorylated p-GlcNAc derivatives of the invention may be prepared,for example, by utilizing techniques well known to those of skill in theart, such as those described by Nishi et al. (Nishi, N. et al., 1986, in“Chitin in Nature and Technology, Muzzarelli et al., eds. Plenum Press,New York, pp. 297-299). Briefly, p-GlcNAc/methanesulfonic acid mixturemay be treated with phosphorus pentoxide (in an approximately 0.5 to 4.0molar equivalent) with stirring, at a temperature of about 0° C. toabout 5° C. Treatment may be for about 2 hours. The resulting productmay then be precipitated and washed using standard techniques well knownto those of skill in the art. For example, the sample may beprecipitated with a solvent such as ether, centrifuged, washed with asolvent such as ether, acetone, or methanol, and dried.

Nitrated p-GlcNAc derivatives may be prepared by utilizing techniqueswell known to those of skill in the art, such as those described bySchorigin and Halt (Schorigin, R. and Halt, E., 1934, Chem. Ber.67:1712). Briefly, p-GlcNAc and/or a p-GlcNAc derivative may be treatedwith concentrated nitric acid to form a stable nitrated product.

One or more of the monosaccharide units of the p-GlcNAc of the inventionmay contain a sulfonyl group, as depicted below:

where R₃ may be an alkyl, an aryl, an alkenyl, or an alkynyl moiety.Such a derivative may be generated by well known methods such as themethod described in Kurita et al. (Kurita, K. et al., 1990, Polym. Prep[Am. Chem. Soc., Div. Polym. Chem.] 31:624-625). Briefly, an aqueousalkali p-GlcNAc solution may be reacted with a chloroform solution oftosyl chloride, and the reaction may then be allowed to proceed smoothlyat low temperatures.

One or more of the monosaccharides of the p-GlcNAc of the invention orits deacetylated derivative may contain one or more O-acyl groups, asdepicted below:

where R₄ and/or R₅, in place of hydrogen, may be an alkyl, an alkenyl,or an alkynyl moiety, and R₆ may be an alkyl, an alkenyl, or an alkynylmoiety. An example of such a derivative may be generated by well knownmethods such as those described by Komai (Komai, T. et al., 1986, in“Chitin in Nature and Technology”, Muzzarelli et al., eds., PlenumPress, New York, pp. 497-506). Briefly, p-GlcNAc may be reacted with anyof a number of suitable acyl chlorides in methanesulfonic acid to yieldp-GlcNAc derivatives which include, but are not limited to, caproyl,capryl, lanoyl, or benzoyl derivatives.

One or more of the monosaccharides of the deacetylated p-GlcNAc of theinvention may contain an N-acyl group, as depicted below:

where R₇ may be an alkyl, an alkenyl, or an alkynyl moiety. Such aderivatization may be obtained by utilizing techniques well known tothose of skill in the art, such as the technique described in Hirano etal. (Hirano, S. et al., 1976, Carbohydrate Research 47:315-320).

Deacetylated p-GlcNAc is soluble in a number of aqueous solutions oforganic acids. The addition of selected carboxylic anhydrides to suchp-GlcNAc-containing solutions, in aqueous methanolic acetic acid,results in the formation of N-acyl p-GlcNAc derivatives.

One or more of the monosaccharides of the deacetylated p-GlcNAc of theinvention or of its deacetylated derivative, may contain an O-alkylgroup, as depicted below:

where R₈ may be an alkyl, and alkenyl, or a alkynyl moiety. Such aderivatization may be obtained by using techniques well known to thoseof skill in the art. For example, the procedure described by Maresh etal. (Maresh, G. et al., in “Chitin and Chitosan,” Skjak-Braek, G. etal., eds., 1989, Elsevier Publishing Co., pp. 389-395). Briefly,deacetylated p-GlcNAc may be dispersed in dimethoxyethane (DME) andreacted with an excess of propylene oxide. The period of the reactionmay be 24 hours, and the reaction takes place in an autoclave at 40 to90° C. The mixture may then be diluted with water and filtered. The DMEmay be removed by distillation. Finally, the end-product may be isolatedvia lyophilization.

One or more of the monosaccharide units of the p-GlcNAc of the inventionmay be an alkali derivative, as depicted below:

Such a derivative may be obtained by using techniques well known tothose of skill in the art. For example, a method such as that describedby Noguchi et al. (Noguchi, J. et al., 1969, Kogyo Kagaku Zasshi72:796-799) may be utilized. Briefly, p-GlcNAc may be steeped, undervacuo, in NaOH (43%, preferably) for a period of approximately two hoursat about 0° C. Excess NaOH may then be removed by, for example,centrifugation in a basket centrifuge and by mechanical pressing.

One or more of the monosaccharide units of the deacetylated derivativeof the p-GlcNAc of the invention may contain an N-alkyl group, asdepicted below:

where R₉ may be an alkyl, an alkenyl, or an alkynyl moiety. Such aderivatization may be obtained by utilizing, for example, a proceduresuch as that of Maresh et al. (Maresh, G. et al., in “Chitin andChitosan,” Skjak-Brack, G. et al., eds. 1989, Elsevier Publishing Co.,pp. 389-395), as described, above, for the production of O-alkylp-GlcNAc derivatives.

One or more of the monosaccharide units of the deacetylated derivativeof the p-GlcNAc of the invention may contain at least one deoxyhalogenderivative, as depicted below:

where R₁₀ may be F, Cl, Br, or I, with I being preferred. Such aderivative may be obtained by using techniques well known to those ofskill in the art. For example, a procedure such as that described byKurita et al. (Kurita, K. et al., 1990, Polym. Prep. [Am. Chem. Soc.Div. Polym. Chem.] 31:624-625) may be utilized. Briefly, a tosylatedp-GlcNAc is made to react with a sodium halide in dimethylsulfoxide,yielding a deoxyhalogen derivative. p-GlcNAc tosylation may be performedby reacting an aqueous alkali p-GlcNAc solution with a chloroformsolution of tosyl chloride. Such a reaction may proceed smoothly at lowtemperatures.

One or more of the monosaccharide units of the deacetylated derivativeof the p-GlcNAc of the invention may form a salt, as depicted below:

where R₁₁, may be an alkyl, an alkenyl, or an alkynyl moiety. Such aderivatization may be obtained by using techniques well known to thoseof skill in the art. For example, a procedure such as that described byAustin and Sennett (Austin, P. R. and Sennett, S., in “Chitin in Natureand Technology,” 1986, Muzzarelli, R. A. A. et al., eds. Plenum Press,pp. 279-286) may be utilized. Briefly, deacetylated p-GlcNAc may besuspended in an organic medium such as, for example, ethyl acetate orisopropanol, to which may be added an appropriate organic acid such as,for example, formic, acetic, glycolic, or lactic acid. The mixture maybe allowed to stand for a period of time (1 to 3 hours, for example).The temperature of reaction and drying may vary from about 12° to about35° C., with 20° to 25° C. being preferred. The salts may then beseparated by filtration, washed with fresh medium, and the residualmedium evaporated.

One or more of the monosaccharide units of the deacetylated derivativeof the p-GlcNAc of the invention may form a metal chelate, as depictedbelow:

where R₁₂ may be a metal ion, particularly one of the transition metals,and X is the dative bond established by the nitrogen electrons presentin the amino and substituted amino groups present in the deacetylatedp-GlcNAc.

One or more of the monosaccharide units of the deacetylated derivativeof the p-GlcNAc of the invention may contain an N-alkylidene or anN-arylidene group, as depicted below:

where R₁₃ may be an alkyl, an alkenyl, an alkynyl, or an aryl moiety.Such a derivatization may be obtained by using techniques well known tothose of skill in the art. For example, a procedure such as thatdescribed by Hirano et al. (Hirano, S. et al., 1981, J. Biomed. Mat.Res. 15:903-911) may be utilized. Briefly, an N-substitution reaction ofdeacetylated p-GlcNAc may be performed with carboxylic anhydrides and/orarylaldehydes to yield acyl- and/or arylidene derivatives.

Further, the p-GlcNAc of the invention, or its deacetylated derivative,may be subjected to controlled hydrolysis conditions, which yield groupsof molecules having uniform, discrete molecular weight and otherphysical characteristics. Such hydrolysis conditions may include, forexample, treatment with the enzyme, lysozyme. p-GlcNAc may be exposed tolysozyme for varying periods of time, in order to control the extent ofhydrolysis. In addition, the rate of hydrolysis may be controlled as afunction of the extent to which the p-GlcNAc that is being lysozymetreated has been deacetylated. Deacetylation conditions may be asdescribed earlier in this Section. The more fully a p-GlcNAc moleculehas been deacetylated, between about 20 and about 90 percentdeacetylated, the more fully the molecule will be hydrolyzed in a giventime. Changes in physical characteristics, in addition to the loweringof molecular weight, may be elicited by hydrolysis and/or deacetylationtreatments. Extensive hydrolysis causes liquefication of the p-GlcNAc.The results of a hydrolysis/deacetylation procedure are presented belowin the Working Example of Section 9, below.

Further, heat denaturation may function to modify the crystallinestructure of the p-GlcNAc. Such a modification of the p-GlcNAc productcrystalline structure may advantageously affect, for example, thereactivity of the p-GlcNAc.

Further, a variety of molecules may be covalently or non-covalentlyfunctionally attached to the deacetylated derivatives of the p-GlcNAc ofthe invention. Such molecules may include, but are not limited to suchpolypeptides as growth factors, such as nerve growth factor, proteases,such as pepsin, hormones, or peptide recognition sequences such as RGDsequences, fibronectin recognition sequences, laminin, integrins, celladhesion molecules, and the like. See, e.g., the compounds discussed,below, in Section 5.6.1.1. Covalent attachment of molecules to theexposed primary amines of deacetylated p-GlcNAc may be accomplished by,for example, chemical attachment utilizing bi-functional cross-linkingreagents that act as specific length chemical spacers. Such techniquesare well known to those of skill in the art, and may resemble, forexample, the methods of Davis and Preston (Davis, M. and Preston, J. F.1981, Anal. Biochem. 116:404-407) and Staros et al. (Staros, J. V. etal., 1986, Anal. Biochem. 156:220-222). Briefly, carboxylic residues onthe peptide to be attached to the deacetylated or partially deacetylatedp-GlcNAc of the invention may be activated and then crosslinked to thep-GlcNAc. Activation may be accomplished, for example, by the additionof a solution such as carbodiimide EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) to a peptide solution ina phosphate buffer. Preferably, this solution would additionally containa reagent such as sulpho-NHS (N-hydroxysulphosuccinimide) to enhancecoupling. The activated peptide may be crosslinked to the deacetylatedp-GlcNAc by mixing in a high pH buffer, such as carbonate buffer (pH9.0-9.2).

The biological activity of the attached peptide (or any covalentlyattached molecule) can be maintained by varying the length of the linkermolecule (e.g., the bi-functional crosslinking compound) utilized toattach the molecule to the

p-GlcNAc of the invention. An appropriate linker length for a givenmolecule to be attached which will not alter the biological activity ofthe attached molecule can routinely be ascertained. For example, thebiological, activity (e.g., a therapeutically effective level ofbiological activity) of a molecule which has been attached via a linkerof a given length can be tested by utilizing well-known assays specificfor the given molecule being attached.

Additionally, in order to maintain the biological activity of themolecule being attached, it may be necessary to utilize a linker whichcan be cleaved by an appropriate naturally occurring enzyme to releasethe peptide (or any covalently attached molecule).

As above, assays commonly employed by those of skill in the art may beused to test for the retention of the biological activity of theparticular molecule being attached to ensure that an acceptable level ofactivity (e.g., a therapeutically effective level activity) is retained.

Alternatively, molecules such as those described above may benon-covalently attached to p-GlcNAc and its derivatives using techniqueswell known to those of skill in the art. For example, a molecule ormolecules of choice may be mixed with suspensions of p-GlcNAc, withdeacetylated or partially deacetylated p-GlcNAc solution, with ap-GlcNAc-lactate solution, with a deacetylated or partially deacetylatedp-GlcNAc salt solution, or with any p-GlcNAc derivative solution. Themixtures can then be lyophilized. Molecules become bound to the p-GlcNAcmatrices following lyophilization, presumably via hydrophobic,electrostatic and other non-covalent interactions. Such p-GlcNAcformulations are, therefore, very easy to produce. Further, suchformulations can effectively be achieved with a wide variety ofmolecules having a broad spectrum of physical characteristics and watersolubility properties, ranging from the most hydrophobic to the mosthydrophilic. Upon attachment of the molecule or molecules, assayscommonly employed by those of skill in the art to test the activity ofthe particular non-covalently attached molecule or molecules can be usedto ensure that an acceptable level of activity (e.g., a therapeuticallyeffective activity) is achieved with the attached molecule.

Encapsulation using the p-GlcNAc of the invention may be achieved usingmethods known in the art. For example, one method for achieving thep-GlcNAc encapsulation can involve the procedure outlined by Hwang etal. (Hwang, C. et al. in Muzzarelli, R. et al., eds., 1985, “Chitin inNature and Technology”, Plenum Press, pp. 389-396) which is incorporatedby reference in its entirety.

Encapsulation can also be achieved, for example, by following amodification of the acid treatment/neutralization variation of thechemical/biological purification method presented, above, in Section5.3.2. Rather than raising the pH of the p-GlcNAc solution toapproximately neutral pH range (i.e., approximately 7.4), one may createa basic pH environment, by raising the pH to approximately 9.0 after thepurification of the p-GlcNAc is completed. At a more basic pH, thestructure of the p-GlcNAc of the invention, or a derivative thereof,assumes a more three-dimensional or “open” configuration. As the pH islowered, the molecule's configuration reverts to a more compact,“closed” configuration. Thus, a compound or drug of interest may beadded to a p-GlcNAc at a high pH, then the pH of the p-GlcNAc/drugsuspension may be lowered, thereby “trapping” or encapsulating the drugof interest within a p-GlcNAc matrix.

Alternatively, hybrids comprising p-GlcNAc and/or p-GlcNAc derivativesmay be formed. Such hybrids may contain any of a number of naturaland/or synthetic materials, in addition to p-GlcNAc and/or p-GlcNAcderivatives. For example, hybrids may be formed of p-GlcNAc and/orp-GlcNAc derivatives plus one or more extracellular matrix (ECM)components. Such ECM components may include, but are not limited to,collagen, fibronectin, glycosaminoglycans, and/or peptidoglycans.Hybrids may also be formed of p-GlcNAc and/or p-GlcNAc derivatives plusone or more synthetic materials such as, for example, polyethylene. Sucha p-GlcNac/polyethylene or p-GlcNac derivative/polyethylene hybrid maybe made by thermally linking the hybrid components via, for example,autoclaving.

In the case of a collagen/p-GlcNAc hybrid, briefly, a p-GlcNAcsuspension and a collagen suspension may be mixed and lyophilized, andcrosslinked, preferably dehydrothermally crosslinked. The collagenspecies of such hybrids may be native or synthetic, and may be of humanor non-human, such as bovine, for example, origin. p-GlcNAc/collagenand/or p-GlcNAc derivative/collagen hybrid materials exhibit uniformproperties, and form a porous matrix that may act, for example, as anefficient three-dimensional matrix for the attachment and growth ofcells. The Working Example presented in Section 13, below demonstratesthe formation, properties and usefulness of such a p-GlcNAc/collagenhybrid.

Additionally, an iodo-p-GlcNAc derivative may be copolymerized with, forexample, styrene, for the manufacture of novel plastic materials.Iodo-p-GlcNAc can be prepared by a process similar to that described byKurita and Inoue (Kurita, K. and Inoue, S., 1989, in “Chitin andChitosan”, Skjak-Braek et al., eds., Elsevier Science Publishing Co.,Inc., p. 365), via tosylation and iodination of p-GlcNAc. The iododerivative of p-GlcNAc can then be dispersed in nitrobenzene and reactedwith styrene, with tin (IV) chloride being used as a catalyst.

Hybrids comprising combinations of deacetylated p-GlcNAc and suchcompounds as, for example, heparin, sodium alginate, and carboxymethylp-GlcNAc may be formulated using techniques such as those describedherein. Such combinations may be formed or reformed into, for example,membranes and fibers.

Complexes of deacetylated p-GlcNAc with polyanions such as, for example,polyacrylic acid or pectin, possessing both positive and negativecharges, may be formulated. The formation of such complexes may beaccomplished according to a method similar to that described by Mireleset al. (Mireles, C. et al., 1992, in “Advances in Chitin and Chitosan”,Brine, C. J. et al., eds., Elsevier Publishers, Ltd.). Deacetylatedp-GlcNAc and polyacrylic acid, carrageenan or pectin, for example, aredissolved in HCl and NaCl, respectively, and the reactant solutions,with equal pH, are mixed. This operation produces effective moleculespossessing both positive and negative characteristics, useful, forexample, in the immobilization of enzymes and therapeutic compounds.

Further, derivatives, such as partially deacetylated derivatives andcarboxymethyl derivatives of the p-GlcNAc of the invention, can be usedto coat liposomes to give them greater stability. Liposomes areartificially constructed microspheres of lipid bilayer useful, forexample, for drug delivery. The compositions of present invention can beused to form negatively charged phospholipids bound to positivelycharged deacetylated p-GlcNAc enclosing an aqueous compartment. Theseconstructions are designed to contain and deliver pharmaceuticals orother components more efficiently than would otherwise be possible byway of oral or other application. Such constructions can be produced byutilizing the pGlcNAc compositions of the invention in conjunction withmethods well known in the art. See, for example, the Dong and Rogers(Dong, C. and Rogers, J. A., 1991, Journal of Controlled Release,17:217-224) which is incorporated by reference in its entirety.

Certain derivatizations of the p-GlcNAc of the invention, or of itsderivatives, may be preferred for specific applications, which aredescribed in Section 5.6, below. For example, sulfated, phosphorylated,and/or nitrated p-GlcNAc derivatives may be preferred as anticoagulantsor as lipoprotein lipase activators. N-acyl p-GlcNAc derivatives mayalso be preferred for anticoagulants, in addition to being preferredfor, for example, use in production of artificial blood vessels,anti-viral compounds, anti-tumor (specifically, cancer cell aggregatingcompounds), dialysis and ultrafiltration membranes, and in theproduction of controlled release drug delivery systems. O-alkyl p-GlcNAcand its deacetylated derivatives may also be preferred in the productionof controlled release drug delivery systems. N-alkyl p-GlcNAcderivatives may be preferred as anti-bacterial agents. Oxido deaminatedderivatives may be preferred as anti-cancer agents, specifically theiruse in conjunction with immunotherapy for cancer cells. Deacetylatedp-GlcNAc derivatives may be preferred as wound healing agents.N-alkylidene and N-arylidene p-GlcNAc derivatives may be preferred forthe enzyme immobilization applications.

5.5. Reformulations

The p-GlcNAc of the invention, as well as its deacetylated derivativesand/or their derivatizations, such as those described, above, in Section5.4, may be dissolved and subsequently reformulated into a variety ofshapes and configurations.

Solution of the p-GlcNAc of the invention can be achieved by treatmentwith dimethyl acetamide (DMA)/lithium chloride. p-GlcNAc may be readilydissolved by stirring in a DMA solution containing 5% LiCl (by weight ofthe DLMA). Water soluble p-GlcNAc derivatives, such as p-GlcNAc salts,may be dissolved in water. P-GlcNAc which has been at least about 75%deacetylated may be put into solution in, for example, a mild acidicsolution, such as 1% acetic acid. p-GlcNAc derivatives that arewater-insoluble may be put into solution in organic solvents.

Derivatization of p-GlcNAc in DMA:LiCl with phenyl isocyanates may beused to produce carbanilates. Further, derivatization of p-GlcNAc inDMA:LiCl with toluene-p-sulphonylchloride may be used to producetoluene-p-sulfonate.

The p-GlcNAc of the invention, its deacetylated derivatives, and/ortheir derivatizations in solution may then be precipitated andreformulated into shapes which include, but are not limited to, mats,strings, microspheres, microbeads, membranes, fibers, powders, andsponges. Further, ultrathin (i.e., less than about 1 micron thick)uniform membranes may be formulated. Additionally, pharmaceuticalformulations such as pills, tablets and capsules can be prepared.

Such reformulations may be achieved, by, for example, taking advantageof the fact that pure p-GlcNAc is insoluble in solutions such as waterand alcohol, preferably ethanol. Introduction, by conventional means,such as by injection, for example, of the p-GlcNAc-containing DMA/LiClmixture into such a water or alcohol, preferably ethanol, solution willbring about the reprecipitation, and therefore reformulation, of thedissolved p-GlcNAc. Such a pure p-GlcNAc reformulation is demonstratedin the Working Example presented, below, in Section 11. In the case ofwater soluble p-GlcNAc derivatives, reformulations may be achieved byreprecipitating in such organic solvents as, for example, ethyl acetateor isopropanol. Reformulations of p-GlcNAc which has been at least about75% deacetylated may be achieved by reprecipitating in an alkalinesolution. Water-insoluble p-GlcNAc derivatives may be reformulated byreprecipitation in aqueous solutions, such as, for example, water.

Deacetylated p-GlcNAc, in conjunction with oxidized cotton, may beformulated to produce p-GlcNAc/cotton hybrid materials improving thewet-strength of paper products. An oxidized cotton substrate can beapproached closely by the deacetylated p-GlcNAc chain which has a flatribbon-like shape, similar to that of cellulose. Such proximitymaximizes the contribution of the van der Waals forces to the forcespromoting adsorption, thus enhancing the wet-strength properties of thehybrid p-GlcNAc-cellulose materials.

p-GlcNAc membranes and three-dimensional p-GlcNAc matrices may beproduced via methods which provide for the formation of controlledaverage pore sizes within either the membranes or the matrices. Poresize can be controlled in membranes and matrices by varying the amountof p-GlcNAc material used, and by the addition of certain solvents suchas methanol or ethanol, with ethanol being preferred, in specificamounts, ranging from about 5% to about 40%, prior to the formation ofmembranes and/or matrices. In general, the greater the percentage ofsolvent, the smaller the average pore size formed will be. The Examplepresented, below, in Section 15, demonstrates the synthesis andcharacterization of such porous p-GlcNAc structures.

Thus, the p-GlcNAc may be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thep-GlcNAc may be used in place of, or in addition to, the excipients andfillers. Tablets may be coated using p-GlcNAc using methods well knownin the art. Liquid preparations for oral administration may take theform of, for example, solutions, syrups or suspensions, or they may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol or fractionated vegetable oils); and preservatives (e.g., methylor propyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate.

5.6. Uses

The p-GlcNAc of the invention, as well as its deacetylated derivativesand their derivatizations, such as those described, above, in Section5.4, and reformulations, such as those described above, in Section 5.5,have a variety of uses. For example, the non-toxic, non-pyrogenic,biodegradable, and biocompatible properties of the molecules of theinvention, in addition to the advantageous properties of the p-GlcNAcand its derivatives, as described herein, lend themselves toapplications in such diverse fields as agriculture, cosmetics, thebiomedical industry, animal nutrition and health, and the food,chemical, photographic, and pharmaceutical industries.

5.6.1. Biomedical Uses of p-GlcNAc Materials 5.6.1.1. DrugImmobilization/Delivery Uses

Biomedical uses of p-GlcNAc material may include, for example, enzymeand/or drug immobilization/delivery methods. The p-GlcNAc/drugformulations of the invention can provide additional benefits to theknown drug formulations, including, for example, increasedeffectiveness, reduced toxicity and improved bioavailability. SuchpGlcNAc/drug formulations can act as therapeutic agents which mayinclude, but are not limited to, antitumor agents, antibiotics,antibacterials, antifungals, anti-virals and anti-inflammatory drugs.

The drug/p-GlcNAc compositions can be formulated, for example, byimmobilizing a given drug by covalent or non-covalent attachment to, thep-GlcNAc or p-GlcNAc derivates of the invention. Techniques for covalentor non-covalent attachment are well known to those skilled in the art,and may be as described. The drug/p-GlcNAc compositions of the inventioncan also be formulated, for example, by encapsulating a given moleculewithin p-GlcNAc or an p-GlcNAc derivative. Encapsulation methods aredescribed, above, in Section 5.4.

Upon attachment or encapsulation of the molecule, assays commonlyemployed by those of skill in the art may be utilized to test theactivity of the particular molecule or molecules attached, therebyensuring that an acceptable level of biological activity (e.g., atherapeutically effective activity) is retained by the attached moleculeor encapsulated molecule.

p-GlcNAc/drug formulations and p-GlcNAc/drug encapsulations may bedelivered to a patient via a variety of routes using standard procedureswell known to those of skill in the art. For example, such delivery maybe site-specific, oral, nasal, intravenous, subcutaneous, intradermal,transdermal, intramuscular or intraperitoneal administration. Bothp-GlcNAc/drug formulations and p-GlcNAc/drug encapsulations may beformulated to function as controlled, slow release vehicles, asdescribed in this Section, below.

With respect to site-specific delivery, administration methods mayinclude, but are not limited to injection, implantation, arthroscopic,laparoscopic or similar means. p-GlcNAc membranes and/or gels as well asmicrospheres and sponges are preferred for such site-specific deliverymethods.

There are numerous advantages in using the p-GlcNAc based drug deliverysystems of the invention. Traditional drug administration by injectionis commonly used with is proteins and many other drugs. However,repeated doses lead to oscillating blood drug concentrations and affectpatient comfort and compliance. Oral administration can be advantageoussince it allows for a more varied load of the drug to be released and isless discomforting to the patient. However, proteins and other compoundsare denatured and degraded in the stomach.

An improved oral administration, however, is achieved by thecompound-containing p-GlcNAc molecules or p-GlcNAc compoundencapsulations of the invention by providing a protective environmentfor the drug once administered. For example, the p-GlcNAc of theinvention protects a compound, such as a protein, from the acidic andenzymatic environment of the stomach. The p-GlcNAc system releases thecompound via diffusion and/or encapsulation degradation once it reachesthe intestinal region, where it is effectively absorbed into the bloodstream. These p-GlcNAc systems of the invention can be used, forexample, to deliver proteins, such as, insulin, as well as many othercompounds. Liposomes coated with p-GlcNAc derivatives or p-GlcNAcderivatives-alginate encapsulations are preferred for such oral deliverymethods.

Upon introduction of the compound-containing p-GlcNAc and/or p-GlcNAcencapsulations into a patient, the p-GlcNAc of the inventionbiodegrades, such that the attached compounds are gradually releasedinto the bloodstream of the patient, thus providing a method forcontrolled compound or drug delivery.

Deacetylated or partially deacetylated p-GlcNAc species may be producedhaving a predictable rate of biodegradability. For example, thepercentage of deacetylation affects the rate at which the p-GlcNAcspecies degrades. Generally, the higher the percentage of deacetylation,the faster the rate of biodegradability and resorption will be. Thus,the degree of p-GlcNAc biodegradability and the in vivo rate ofresorption may be controlled during the p-GlcNAc's production. Examplesof the production and characterization of such p-GlcNAc materials arepresented in Section 18, below. p-GlcNAc materials having suchcontrollable biodegradability rates may be formulated into membranes,gels, sponges, microspheres, fibers, and the like. These p-GlcNAcproducts adhere and mold to tissues, both soft and hard tissues, in thehuman body with no need for suturing. The p-GlcNAc materials may, forexample, be applied during general or minimally invasive surgery, suchas laparoscopic surgery.

Compound-p-GlcNAc and p-GlcNAc encapsulations have a variety ofapplications as therapeutic drug delivery systems. Compounds which maybe encapsulated within or attached to (covalently or non-covalently) thep-GlcNAc, or a derivative of the invention are, for example, antitumorcompounds, antibiotics, antibacterials, antifungals, antivirals, smallpeptide and non-peptide molecules, vitamins and other health-relatedfood additives. Anti-tumor drugs which can be attached to, orencapsulated within the p-GlcNAc of the invention include, but are notlimited to, those listed in this Section, below.

Additionally, combinations of two or more molecules may be encapsulatedwithin or attached to the p-GlcNAc of the invention to provide asynergistic effect. For example, anti-tumor agents such as thioguaninecombined with cytosine arabinoside (ara-C) are contemplated for use inthe invention as an improved treatment for acute nonlymphocyticleukemia. Other synergistic combinations include tamoxifen withcisplatin for breast cancer, and prostaglandins with cisplatin forbreast and prostate cancer. Additionally, many other synergisticcombinations of anti-cancer drugs, known to those of skill in the art,may be used with the p-GlcNAc and p-GlcNAc derivative formulations ofthe invention.

Further, gene therapy agents, such as anti-sense DNA and ribozymesystems may also be encapsulated within the p-GlcNAc or p-GlcNAcderivatives of the invention. Immunotherapeutic compounds (e.g.,vaccines), such as tumor specific antigens, that can elicit an immuneresponse (e.g., a cytotoxic T-lymphocyte response) against a specifictumor type (e.g., melanoma) can also be attached to, or encapsulatedwithin the p-GlcNAc of p-GlcNAc derivatives of the invention by methodsknown in the art.

The drug delivery systems described herein are feasible for use with anyanti-tumor drug. For example, the use of such anti-tumor drug deliverysystems is demonstrated in the Example presented in Section 20, below.Such drugs are well known to those of skill in the art, and may beformulated into p-GlcNAc gels or membranes, for example, so as toprovide site-specific slow-release delivery directly to the tumor or tothe region vacated by the tumor following surgery. Such an immobilizedslow-release p-GlcNAc drug product can act as an important initialdefensive procedure after surgery. Such p-GlcNAc anti-tumor drugdelivery systems are particularly useful in treating tumors which aretotally or partially inaccessible through surgery, such as, for example,is the case with certain brain tumors.

Additionally, the use of p-GlcNAc/compound and p-GlcNAc encapsulationsof the invention for the development of new anti-tumor drug formulationsis desirable given that the p-GlcNAc polymer has chemical properties andcharacteristics making possible the formulation and delivery of somedrugs that have heretofore been difficult to formulate and deliver. Forexample, taxol, a microtubule spindle inhibitor drug used to treatbreast cancer, is hydrophobic and requires the addition ofpolyoxyethylated castor oil in order to solubilize it as a liquidinfusion for intravenous delivery. The hydrophobic nature of taxol makesit an ideal compound for formulation with p-GlcNAc polymer materials fortopical controlled release delivery. The Example presented in Section23, below, presents such a p-GlcNAc/taxol formulation. Additionaltargets for p-GlcNAc anti-tumor systems include, but are not limited to,skin, GI tract, pancreatic, lung, breast, urinary tract and uterinetumors, and HIV-related Kaposi's sarcomas.

For example, anti-tumor drugs that may be formulated with the p-GlcNAcand p-GlcNAc encapsulation system of the invention include, but are notlimited to, the following categories and specific compounds alkylatingagents, antimetabolite agents, anti-tumor antibiotics, vinea alkaloidand epidophyllotoxin agents, nitrosoureas, enzymes, synthetics, hormonaltherapeutic biologics and investigational drugs.

Such alkylating agents may include, but are not limited to nitrogenmustard, chlorambucil, cyclophosphamide, ifosfamide, melphalan, thiptepaand busulfan.

Antimetabolites can include, but are not limited to, methotrexate,5-fluorouracil, cytosine arabinoside (Ara-C), 5-azacytidine,6-mercaptopurine, 6-thioguanine, and fludarabine phosphate. Antitumorantibiotics may include but are not limited to doxorubicin,daunorubicin, dactinomycin, bleomycin, mitomycin C, plicamycin,idarubicin, and mitoxantrone. Vinca alkaloids and epipodophyllotoxinsmay include, but are not limited to vincristine, vinblastine, vindesine,etoposide, and teniposide.

Nitrosoureas such as, but not limited to carmustine, lomustine,semustine and streptozocin. Enzymes can include, but are not limited toL-asparagine.

Synthetics can include, but are not limited to Dacrabazine,hexamethylmelamine, hydroxyurea, mitotane procabazine, cisplatin andcarboplatin.

Hormonal therapeutics can include, but are not limited tocorticosteriods (cortisone acetate, hydrocortisone, prednisone,prednisolone, methyl prednisolone and dexamethasone), estrogens,(diethylstibesterol, estradiol, esterified estrogens, conjugatedestrogen, chlorotiasnene), progestins (medroxyprogesterone acetate,hydroxy progesterone caproate, megestrol acetate), antiestrogens(tamoxifen), aromastase inhibitors (aminoglutethimide), androgens(testosterone propionate, methyltestosterone, fluoxymesterone,testolactone), antiandrdgens (flutamide), LHRH analogues (leuprolideacetate), and endocrines for prostate cancer (ketoconazole).

Biologics can include, but are not limited to interferons, interleukins,tumor necrosis factor, and biological response modifiers.

Investigational Drugs can include, but are not limited to alkylatingagents such as Nimustine AZQ, BZQ, cyclodisone, DADAG, CB10-227, CY233,DABIS maleate, EDMN, Fotemustine, Hepsulfam, Hexamethylmelamine,Mafosamide, MDMS, PCNU, Spiromustine, TA-077, TCNU and Temozolomide;antimetabolites, such as acivicin, Azacytidine, 5-aza-deoxycytidine,A-TDA, Benzylidene glucose, Carbetimer, CB3717, Deazaguanine mesylate,DODOX, Doxifluridine, DUP-785, 10-EDAM, Fazarabine, Fludarabine, MZPES,MMPR, PALA, PLAC, TCAR, TMQ, TNC-P and Piritrexim; antitumor antibodies,such as AMPAS, BWA770U, BWA773U, BWA502U, Amonafide, m-AMSA, CI-921,Datelliptium, Mitonafide, Piroxantrone, Aclarubicin, Cytorhodin,Epirubicin, esorubicin, Idarubicin, Iodo-doxorubicin, Marcellomycin,Menaril, Morpholino anthracyclines, Pirarubicin, and SM-5887;microtubule spindle inhibitors, such as Amphethinile, Navelbine, andTaxol; the alkyl-lysophospholipids, such as BM41-440, ET-18-OCH₃, andHexacyclophosphocholine; metallic compounds, such as Gallium itrate,CL286558, CL287110, Cycloplatam, DWA2114R, NK121, Iproplatin,Oxaliplatin, Spiroplatin, Spirogermanium, and Titanium compounds; andnovel compounds such as, for example, Aphidoicolin glycinate, Ambazone,BSO, Caracemide, DSG, Didemnin, B, DMFO, Elsamicin, Espertatrucin,Flavone acetic acid, HMBA, HHT, ICRF-187, Iododeoxyuridine, Ipomeanol,Liblomycin, Lonidamine, LY186641, MAP, MTQ, Merabarone SK&F104864,Suramin, Tallysomycin, Teniposide, THU and WR2721; and Toremifene,Trilosane, and zindoxifene.

Antitumor drugs that are radiation enhancers are preferred for instancesin which radiation therapy treatment is to be prescribed, either in lieuof, or following surgery. Examples of such drugs include, for example,the chemotherapeutic agents 5′-fluorouracil, mitomycin, cis-platin andits derivatives, taxol, doxorubicin, actinomycin, bleomycins,daunonycins, and methamycins.

Dose ranges for anti-tumor drugs may be lower than, equal to or greaterthan the typical daily doses prescribed for systemic treatment ofpatients. Higher doses may be tolerated in that the drugs are deliveredlocally at the site of a tumor, whereas other tissues, therefore,including blood cells, are not as readily exposed to the drugs. Forexample, dosages of 5′-FU equivalent to 50% of the standard dosages usedto treat colorectal cancer with 5′-FU in humans (300-450 mg/m² i.v.daily for 5 days) resulted in an 80-90% reduction in volume of ectopicHT29 colon cancer tumor implants in scid mice. The use of the p-GlcNAcmembrane as a drug delivery matrix for the administration of 5′-FUreduced the dosage required to dramatically reduce tumor volume by 50 ascompared to intravenuous control animals. Details regarding this datacan be found in Example 21, below.

Further, doses of such drugs are well known to those of skill in the artand can be easily found in such compendia as the PHYSICIANS DESKREFERENCE, Medical Economics Data Publishers; REMINGTON'S PHARMACEUTICALSCIENCES, Mack Publishing Co.; GOODMAN & GILMAN, THE PHARMACOLOGICALBASIS OF THERAPEUTICS, McGraw Hill Publishers, THE CHEMOTHERAPY SOURCEBOOK, Williams and Wilkens Publishers, online services such as theCancer Lit®, U.S. National Cancer Institute database, as well as reportsof pharmacological studies such as “A MultiCenter Randomized Trial ofTrial of Two Doses of Taxol” Nabholtz, J. M., Gelmon, K., Bontenbal, M.et al. Medical Education Services Monograph—1994 Bristol-Myers SquibbCompany Publication; “Randomized Trial of Two Doses of taxol inMetastatic Breast Cancer: An Interim Analysis” Nabholtz, J. M., Gelmon,K., Bontenbal, M., et al. 1993, Proc. Am. Clin. Oncol., 12:60. Abstract42 Alternatively, such doses can be routinely determined by usingstandard techniques well known to those of skill in the art such as, forexample, those described, below, at the end of this Section.

Certain anti-tumor agents are vesicants, including dactinomycin,daunomycin, doxorubicin, estramustine, mechlorethamine, mitomycin C,vinblastine, vincristine and videsine; while certain anti-tumor drugsare irritants, including carmustine, decarbazine, etoposide, mithrmycin,streptozocin and teniposide. Vesicants and irritants cause adverseside-effects including extravasation and irritation of tissues withpain, redness, swelling, and other symptoms. Further, tissue necrosiscan result from some of the side effects. The p-GlcNAc membrane and gelmaterials of the invention used for the topical, controlled release ofanti-tumor drugs have wound healing properties. Normal tissues that arein contact with vesicant or irritant anti-tumor drugs delivered by thep-GlcNAc membrane and gel formulations of the invention are, therefore,not as readily damaged and will heal faster due to the active healingeffects of the p-GlcNAc component of the anti-tumor drug-containingp-GlcNAc and p-GlcNAc-drug encapsulations of the invention.

The compound-containing p-GlcNAc and p-GlcNAc-compound encapsulations ofthe invention may, additionally, be used for the treatment ofinfections. For such an application, antibiotics, either water solubleor water insoluble, may be immobilized/formulated in p-GlcNAc basedmaterials, such as, for example, gels and membranes. Antibiotics arewell known to those of skill in the art, and include, for example,penicillins, cephalosporins, tetracyclines, ampicillin, aureothicin,bacitracin, chloramphenicol, cycloserine, erythromycin, gentamicin,gramacidins, kanamycins, neomycins, streptomycins, tobramycin, andvancomycin Doses of such drugs are well known to those of skill in theart, and may, alternatively, routinely be determined using standardtechniques well known to those of skill in the art, such as, forexample, are described, below, at the end of this Section.

Such p-GlcNAc antibiotic products may be used to treat bacterialinfections that occur either externally, e.g., on skin, scalp, dermalulcers or eyes, or internally, e.g., localized infections of the brain,muscles, abdomen. A prominent application is for treatment ofHIV-related opportunistic infections.

The compound-containing p-GlcNAc and p-GlcNAc-compound encapsulationsystems of the invention may be formulated with anti-inflammatory drugsto control dysfunctional activity of the inflammatory and immuneprocesses. For example, p-GlcNAc may be formulated with non-steroidalanti-inflammatory drugs (NSAIDs) and used to the reduction of local painand inflammation induced by diseases such as rheumatoid arthritis,osteoarthritis and systemic lupus, to name a few. The localized deliveryof such NSAIDs using the p-GlcNAc gel or membrane/drug delivery systemsof the invention may serve to reduce NSAID side effects, which mayinclude gastric irritation, azotemia, platelet disfunction and liverfunction abnormalities. NSAIDs are well known to those of skill in theart and include inhibitors of cycloxygenase, such as aspirin, etodolac,fenoprofen and naproxen. Other anti-inflammatory drugs may be utilizedas part of the compound-containing p-GlcNAc and p-GlcNAc encapsulationsystems of the invention, such as, for example, inhibitors of lipidinflammatory mediators, such as leucotrienes. Doses for such drugs arewell known to those of skill in the art, and may, alternatively,routinely be determined using standard techniques well known to those ofskill in the art, such as, for example, are described, below, at the endof this Section.

The compound-containing p-GlcNAc and p-GlcNAc-compound encapsulationsystems of the invention may additionally be formulated with antifungalagents, using techniques described above, for the treatment of specificfungal diseases. Antifungal agents are well known to those of skill inthe art, and may include, for example, amphotericin, anisomycin,antifungone, blastomycin, griseofulvins, and nystatin. Doses of suchdrugs are well known to those of skill in the art, and may,alternatively, routinely be determined using standard techniques wellknown to those of skill in the art, such as, for example, are described,below, at the end of this Section.

The compound-containing p-GlcNAc and p-GlcNAc-compound encapsulationsystems of the invention may also be formulated with antiprotozoalagents, using techniques described above, for the treatment of specificprotozoal infections. Antiprotozoal agents are well known to those ofskill in the art, and may include, for example, antiamoebin,antiprotozin, monomycin, paromomycin and trichomycin. Doses of suchdrugs are well known to those of skill in the art, and may,alternatively, routinely be determined using standard techniques wellknown to those of skill in the art, such as, for example, are described,below, at the end of this Section.

The compound-containing p-GlcNAc and p-GlcNAc-compound encapsulationsystems of the invention may be formulated with spermicidal compounds,using techniques such as those described, above, to produce effectivecontraceptives. Appropriate spermicides are well known to those of skillin the art. Doses of such spermicides are well known to those of skillin the art, and may, alternatively, routinely be determined usingstandard techniques well known to those of skill in the art, such as,for example, are described, below, at the end of this Section.

The compound-containing p-GlcNAc and p-GlcNAc-compound encapsulationsystems of the invention may, still further, be formulated usingtherapeutic protein agents. Such formulations may be produced using, forexample, techniques such as those described above. By utilizing suchp-GlcNAc therapeutic protein systems, it is possible to deliver specificproteins directly to desired target sites and to effect slow release ofthe proteins at such sites. Examples of possible proteins include, butare not limited to insulin, monoclonal antibodies, breast cancerimmunotoxin, tumor necrosis factor, interferons, human growth hormone,lymphokines, colony stimulating factor, interleukins and human serumalbumin. Doses of such therapeutic protein agents are well known tothose of skill in the art and may be found in pharmaceutical compediasuch as the PHYSICIANS DESK REFERENCE, Medical Economics DataPublishers; REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Co.;GOODMAN & GILMAN, THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, McGraw HillPubl., THE CHEMOTHERAPY SOURCE BOOK, Williams and Wilkens Publishers,and may, alteratively, routinely be determined using standard techniqueswell known to those of skill in the art, such as, for example, aredescribed, below, at the end of this Section.

The compound-containing p-GlcNAc and p-GlcNAc-compound encapsulationdelivery systems of the invention can also be formulated as nutritionaland vitamin supplements.

Because the p-GlcNAc materials of the invention are themselvesimmunoneutral, in that they do not elicit an immune response in humans,such p-GlcNAc devices, as described above, comprising p-GlcNAcmembranes, 3D porous matrices and/or gels that harbor immobilized drugs,may deliver such drugs in a manner that there is no immune response.Certain additional materials, such as natural alginates and syntheticpolymers, may be used in some cases to construct such devices incombination with the p-GlcNAc material. For instance, a polymericdelayed-release drug delivery system can be manufactured in a mannersimilar to that suggested by A. Polk (Polk, A. et al., 1994, J. ofPharmaceutical Sciences, 83(2):178-185). In such a procedure,deacetylated p-GlcNAc is reacted with sodium alginate in the presence ofcalcium chloride to form microcapsules containing the drug to bedelivered and released under appropriate conditions and over a certainlapse of time.

The therapeutically effective doses of any of the drugs or agentsdescribed above, in conjunction with the p-GlcNAc-based systemsdescribed herein, may routinely be determined using techniques wellknown to those of skill in the art. A “therapeutically effective” doserefers to that amount of the compound sufficient to result inamelioration of symptoms of the processes and/or diseases describedherein.

Toxicity and therapeutic efficacy of the drugs can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

5.6.1.2. p-GlcNAc Cell Encapsulation Uses

p-GlcNAc encapsulated cells may be formulated, and such p-GlcNAcencapsulated cells may be administered to a patient, via standardtechniques well known to those of skill in the art. See, for example,the techniques described, above, in Section 5.6.1.1. Alternatively, see,for example, Aebisher et al. (Aebisher, P. et al., in “Fundamentals ofAnimal Cell Encapsulation and Immobilization”, 1993, CRC Press, pp.197-224), Yoshioke et al., (Yoshioke, T. et al., 1990, Biotechnol.Bioeng. 35:66) and U.S. Pat. No. 4,749,620, each of which isincorporated herein by reference in its entirety. Cells may beencapsulated by, on, or within p-GlcNAc or partially deacetylatedp-GlcNAc membranes, three-dimensional p-GlcNAc porous matrices, orp-GlcNAc gels.

Three-dimensional matrices can be seeded with cells and used in certainapplications without further encapsulation. Alternatively, cells can beencapsulated into microspheres or droplets of p-GlcNAc-based polymergels such as, for example, a p-GlcNAc-lactate polyelectrolyte polymer (apolycationic polymer). Gels, droplets or microspheres into which cellshave been encapsulated may then be coated with a second polyelectrolyteof opposite charge (e.g., with a polyanion, such as an alginate) to forman outer capsule which provides immuno-isolation for the encapsulatedcells, thus reducing the risk of immune rejection by the host organism.

Additionally, cells entrapped in p-GlcNAc gels, three-dimensionalp-GlcNAc matrices, or both, can be loaded into thermoplastic capsules inyet another method of formulation. Thermoplastic-based capsules can alsobe utilized to provide immuno-protection for implanted cells in a hostorganism. Such thermoplastic capsules are made of materials such ashydroxyethyl methylacrylate-methylmethacrylate copolymer (HEMA-MMA).Thermoplastic-derived microcapsules are formed, for example, by thecoextrusion of a solution of HEMA-MMA in polyethylene glycol and thecell-containing p-GlcNAc matrix and/or gel medium, into an appropriateorganic solvent such as hexadecane. See, for example, the methoddescribed by Aebisher et al. (Aebisher, P. et al., in “Fundamentals ofAnimal Cell Encapsulation and Immobilization”, 1993, CRC Press, pp.197-224).

The p-GlcNAc cell encapsulations have a variety of applications. First,they may be utilized for the delivery of therapeutic compounds,synthesized and secreted by cells attached to and encapsulated in themembranes, matrices or gels.

For example, and not by way of limitation, the p-GlcNAc/cellencapsulations may be used for delivery of insulin in the treatment ofdiabetes, nerve growth factor for the treatment of Alzheimer's disease,factor VIII and other clotting factors for the treatment of hemophilia,dopamine for the treatment of Parkinson's disease, enkephalins viaadrenal chromaffin cells for the treatment of chronic pain, dystrophinfor the treatment of muscular dystrophy, and human growth hormone forthe treatment of abnormal growth. For example, dopamine-producingadrenal chromaffin cells may be encapsulated for the treatment ofParkinson's Disease and for the relief of chronic pain. Also, pancreaticislet cells may be encapsulated for the treatment of insulin-dependentdiabetes mellitus. Further, the p-GlcNAc/cell encapsulations may be usedfor delivery of the above peptides and proteins, or other peptides andproteins, as gene products in vivo for use in gene therapies. Forexample, using recombinant DNA techniques, a gene for which a patient isdeficient could be placed under the control of viral or tissue specificpromotor. The recombinant DNA construct containing the gene could beused to transform or transfect a host cell which is cloned and thenclonally expanded and is engineered so as to secrete the said geneproduct then encapsulated into the p-GlcNAc/cell encapsulations of theinvention and used to deliver the gene product as a therapy.

Additionally, such encapsulated cells may act to compensate for the lossof a specific organ or tissue and/or may be used to augment the reducedfunction of such a specific tissue or organ of the body. For example,encapsulated liver cells may be used to augment reduced liver function,and may, for example, be utilized as a temporary measure prior to theadministration of a liver transplant. As another example, encapsulatedthyroid, parathyroid or pancreas cells may be administered to compensatefor the loss of these glands.

Because the p-GlcNAc materials of the invention are themselvesimmunoneutral, as they do not elicit an immune response in humans, it ispossible to engineer and construct devices consisting of p-GlcNAcmembranes, three-dimensional porous p-GlcNAc matrices and/or p-GlcNAcgels that harbor attached cells which can deliver cell-basedtherapeutics in a manner such that the cells are immuno-isolated, i.e.,no anti-cell host immune response is elicited. Certain additionalmaterials, such as, for example, natural alginates and syntheticpolymers, may be used to construct such devices in addition to thep-GlcNAc material itself.

p-GlcNAc/cell encapsulation compositions may additionally be utilizedfor the delivery of cells to seed tissue regeneration. Applications ofspecific cell types encapsulated for the seeding of cell growth leadingto tissue regeneration at the site of an injury may include, but are notlimited to regeneration of skin, cartilage, nerves, bone, tendon,ligaments, liver and blood vessels. The tissue regeneration applicationsof cells encapsulated in p-GlcNAc materials are advantageous, in part,because of the ability of the p-GlcNAc material to adhere to injuredtissue, to provide a substrate for mammalian cell growth, and to undergobioresorption coincident with the growth of new healthy tissue duringthe tissue regeneration process at the site of injury.

5.6.1.3. Utilizing p-GlcNAc Materials for the Prevention ofPost-Surgical Adhesions

Additionally, p-GlcNAc membranes may be used to provide a biodegradable,biocompatible mechanical barrier to prevent post-surgical adhesions. TheExample presented in Section 17, below, demonstrate such a p-GlcNAcapplication. Solid p-GlcNAc or p-GlcNAc derivatives formulated intomembranes or sponges may be utilized for such an application. Preferredmembranes are thin, generally less than about 1 mm in thickness.Preferable p-GlcNAc derivatives are p-GlcNAc derivatives which have beenabout 50-80% deacetylated. Such p-GlcNAc derivatives will generally beresorbed approximately 7-21 days post-implantation.

Liquid p-GlcNAc derivatives are also suitable for use in the preventionof post-surgical adhesions. Preferable liquid p-GlcNAc derivatives forsuch an application are deacetylated p-GlcNAc salt derivatives andcarboxymethyl p-GlcNAc derivatives. A p-GlcNAc derivative which isparticularly preferred for the prevention of post-surgical adhesions isa p-GlcNAc-lactate derivative, especially a p-GlcNAc-lactate gelderivative. As used herein, the term p-GlcNAc-lactate means that thelactic acid moiety is functionally attached to a partially or fullydeacetylated poly-β-1→4-N-acetylglucosamine of the invention. Suchp-GlcNAc-lactate derivatives may be formulated using propylene glycoland water, as, for example, described in Section 17.1. p-GlcNAc-lactatederivatives may be produced having high and low viscosities, whichallows for the ability to tailor the p-GlcNAc used to the specificindication of interest. For example, it may be useful to use a p-GlcNAcproduct having a lower viscosity for delivery through a syringe or via aspray, while it may be desirable to use a p-GlcNAc product having ahigher viscosity, and therefore greater lubrication properties, when theindication is an orthopedic one.

For the prevention of post-surgical adhesions, solid p-GlcNAcformulations are suitable for clearly circumscribed wound sites. Suchp-GlcNAc formulations should be applied following the surgical procedureand the material should completely cover the traumatized tissue. It canbe applied either in conjunction with either general or minimallyinvasive (e.g., laparoscopic) surgical procedures. The solid p-GlcNAcformulations can be cut and applied using standard surgical proceduresand instrumentation well known to those of skill in the art.

The liquid p-GlcNAc formulations can be applied, for the prevention ofpost-surgical adhesions, in larger areas prone to form suchpostoperative adhesions. The p-GlcNAc-lactate gel, for example, can beapplied before the surgical procedure to provide additional lubricationand thus reduce the amount of traumatized tissue. Alternatively, theliquid p-GlcNAc formulation, such as p-GlcNAc-lactate, can be appliedfollowing the surgical procedure to form a physical barrier to preventpostoperative adhesion formation.

The p-GlcNAc material can be painted, sprayed or dropped from a syringedevice onto the wounded site. In laparoscopic procedures, low viscositymaterials can, for example, be delivered with standard suctionirrigation devices. Higher viscosity materials will require pressure toreach its target. The pressure can be provided by a compressed gaspowered piston or a syringe type device.

The amount of liquid p-GlcNAc formulation, such as the p-GlcNAc-lactategel formulation, required for prevention of post-surgical adhesions isproportional to the extent of the traumatized tissue. The p-GlcNAcmaterial administered should be applied in the range of 0.1 ml to 1.5 mlper sq. cm of surface area.

Various animal models which are known in the art can be used to test thepost-surgical adhesion formulations of the invention. These include, butare not limited to, the Rat cecum model (Harris, E. W., et al., 1992,Journal of Investigative Surgery, 5:260; and the Rabbit uterine hornmodel (Diamond, M. P., et al., 1987, Microsurgery, 8:197.

5.6.1.4. Biodegradable p-GlcNAc Barriers

p-GlcNAc materials having a controllable rate of biodegradation may beuseful as, for example barriers having a variety of applications. Forexample, such barriers may be utilized to promote hemostasis. Thesuccessful use of such a hemostatic p-GlcNAc application is demonstratedin the Example presented, in Section 19, below. Additionally,p-GlcNAc-based material, such as thick gels, sponges, films andmembranes may be used for such hemostatic applications. The p-GlcNAcbased materials, when applied directly to bleeding surfaces, arrestbleeding by providing a mechanical matrix that promotes clotting. Thep-GlcNAc based materials adhere to the site of application and seal theboundaries of the wound. This reduces the amount of blood loss, protectsthe forming clot and facilitates the clotting process.

The preferred solid materials for such an application are deacetylatedp-GlcNAc membranes and sponges. The preferred thick gels can beformulated from water plus soluble derivatives like deacetylatedp-GlcNAc salts and carboxymethyl p-GlcNAc derivatives. The thick gels ofthe invention should have a viscosity of 50,000 cps or greater. Forexample, and not by way of limitation, one formulation of such a gel ispresented in Example 17.1 in formulating p-GlcNAc-lactate as a gel.

Applications for the hemostatic agents described above include, but arenot limited to, uses in diagnostic procedures such as biopsy wounds in,for example, liver and kidney; in trauma wounds, for example, spleen,liver and blood vessel injuries; in standard and minimally invasivesurgical procedures, for example, endometriosis surgery and operationson the gallbladder; in soft and hard tissue wound repair, for example,skin wounds and burn healing; in surgical procedures, in particular, forsplenic wounds; and for blood vessel puncture diagnostic and treatmentprocedures such as catheterization and balloon angioplasty procedures.

The solid p-GlcNAc based materials of the invention can be applied usingstandard surgical procedures, and can be used with both standard andminimally invasive surgical interventions. The thick gels of theinvention can be delivered, for example, by extrusion from a syringetype device or in combination with a membrane or film. The membrane orfilm can be manufactured from a p-GlcNAc based material or other naturalor synthetic materials.

In connection with the blood vessel puncture procedures mentioned above,the hemostatic agents of the invention may be applied at the time when acatheter sheath is being removed from a blood vessel. A device thatdetects the removal of the catheter sheath from the blood vessel can bedeveloped using electronic or mechanical systems that monitor chemical,physical or other differences between the tissue inside and outside ofthe vessel. For example, the differential in fluid dynamics or heatdissipation can be detected when a probe is removed from the vessel; atthat point a signal is sent to initiate the application of thehemostatic agent.

Further, p-GlcNAc materials may be utilized to provide periodontalbarriers for the separation of soft and hard tissue during the repairprocess following periodontal surgery in order to promote uniform tissuerepair, to provide biodegradable contact lenses, corneal shields or bonegrafts, to provide surgical space fillers, to promote soft tissueaugmentation, particularly in the skin for the purpose of reducing skinwrinkles, and as urinary sphincter augmentation, for the purpose ofcontrolling incontinence.

5.6.1.5. Other Biomedical Uses of p-GlcNAc Materials

Other biomedical uses of p-GlcNAc materials include, for example, theuse of such materials as cell culture substrates. For example, as shownin the Working Example presented in Section 12, below, the p-GlcNAc ofthe invention acts as a very efficient substrate for mammalian cellsgrown in culture. Further, three-dimensional configurations of p-GlcNAcmay be used as medium components which will allow three-dimensional cellculture growth.

The cell substrate capabilities of the p-GlcNAc of the invention mayalso be utilized in vivo. Here, the p-GlcNAc of the invention, or aderivative thereof, as described herein, may act to facilitate tissueregeneration (e.g., regeneration of connective tissue covering teethnear the gum line, vascular grafts, ligament, tendon, cartilage, bone,skin, nerve tissues). The p-GlcNAc molecules of the invention may,therefore, for example, have extensive plastic surgery applications.

Deacetylated p-GlcNAc is preferred for use as a sealant of vasculargrafts. Deacetylated p-GlcNAc derivatives such as N-carboxymethyl andN-carboxybutyl deacetylated p-GlcNAc are preferred as tissueregeneration reagents. N-carboxymethyl deacetylated p-GlcNAc may, forexample, be inoculated into the cornea to induce neovascularization.

Further biomedical applications of the p-GlcNAc of the invention or ofits derivatives, as described herein, may involve the molecules, use inwound dressing, wound healing ointments, and surgical sutures, sponges,and the like. Such promotion of wound healing and reduction of scarringis illustrated, below, in examples 17 and 22. These properties areuseful in stimulating tissue repair, and accelerates, strengthen andimprove the quality of healing. p-GlcNAc based materials may be used forinjury related or surgically induced wounds in both soft and hardtissue. For example, such wounds include venous stasis ulcers, burnhealing and surgical wounds in the eye or other tissues where quality ofhealing or fibrosis may be important.

Still further, such molecules may be used, for example, in the treatmentof osteoarthritis, in the reduction of blood serum cholesterol levels,as anti-viral agents, as anti-bacterial agents, as immunomodulators, asanticoagulants, as dialysis and ultrafiltration membranes, as anti-tumoragents, as contact lens material, and as oral adsorbents for uremictoxins when administered to kidney failure patients. Microcrystallinep-GlcNAc suspensions or water soluble p-GlcNAc derivatives are preferredfor the treatment of arthritis, by, for example, injection directly intoarthritic joints.

p-GlcNAc has additional applications as a component of artificial ordonor skin. For example, p-GlcNAc, preferably as non-woven p-GlcNAcfilms, may be applied to split thickness skin donor sites, over, forexample, donor dermis.

Deacetylated p-GlcNAc to which a protease, such as pepsin, has beenattached may be used for the controlled digestion of proteins in contactwith such p-GlcNAc/protease compounds.

5.6.2. Agricultural Uses of p-GlcNAc Materials

The p-GlcNAc of the invention or its derivatives may be used in variousagricultural applications, as well. Such applications include, but arenot limited to insecticide, fungicide, bactericide, and nematocideapplications. N-carboxymethyl deacetylated p-GlcNAc derivatives arepreferred for use as effective bacteriostatic reagents. N-alkyl p-GlcNAcderivatives may be preferred for fungicide applications. Additionally,the molecules of the invention may be used in various soil treatmentapplications, including, but not limited to, fertilizer compositions.Further, controlled release of agrochemicals may be achieved byentrapping such chemicals via the immobilization, encapsulation, andother methods described, above, in this Section. Additionally, analogsof, for example, Rhizobium nodulation factors and/or nitrogen fixationinducers may be immobilized onto, and administered via, the p-GlcNAcand/or p-GlcNAc derivatives of the invention.

5.6.3. Nutrition/Food Industry Uses of p-GlcNAc Materials

The p-GlcNAc of the invention and its derivatives as described hereinadditionally have applications in the fields of animal and humannutrition. For example, the molecules of the invention may be used asfeed ingredients Techniques such as those described, above, in thisSection, may be used in the production of controlled release products inanimal systems. Additionally, the biomedical applications describedabove may be utilized in animal systems by incorporating routinemodifications well known to those of ordinary skill in the art.

Food industry applications of the p-GlcNAc of the invention and of itsderivatives, as described herein, may include, but are not limited toanticholesterol (i.e., hypocholesterolemic compounds), fat-bindingcompounds, emulsifiers, carriers, preservatives, seasonings, and foodtexturizers, in addition to fruit coatings, and food packaging products.For example, in terms of cholesterol and fat binding, p-GlcNAcderivatives exhibit strong binding activity towards lipids. Deacetylatedand appropriately hydrolyzed p-GlcNAc derivatives also bind to lipidsand dietary cholesterol. (Vahouny, G. V., 1983, Journal of ClinicalNutrition 38:278-284). It is possible that p-GlcNAc exerts ahypocholestrolemic effect by forming micelles with cholesterol andlipids, causing them eventually to be excreted (Hirano, S., 1990, in,“The International Symposium on Chitin Derivatives in Life Sciences”)

5.6.4. Cosmetic Uses of p-GlcNAc Materials

Cosmetic applications of the p-GlcNAc of the invention may include, butare not limited to, the production of products for hair and skin care.Skin care products may include, for example, cosmetics utilizingdeacetylated p-GlcNAc salts, carboxymethyl p-GlcNAc-containing products,and cosmetic packs containing deacetylated p-GlcNAc and such derivativesas hydroxypropyl-, N-succinyl-, and quaternary p-GlcNAc derivatives.Hair products may include, for example, carboxymethylp-GlcNAc-containing products, and film-forming p-GlcNAc derivatives.

5.6.5. Chemical Engineering Applications of p-GlcNAc Materials

The p-GlcNAc of the invention and its derivatives have a variety ofapplications that are useful in the chemical engineering industry. Forexample, p-GlcNAc may be used as a coupling agent for adhesion of metalsto polymers, membranes formed by glycol p-GlcNAc may be used indesalination applications, and membranes formed by other p-GlcNAcderivatives may be used for transport of halogen ions. Otherapplications may include the production of flame retardants, and themanufacture of metal chelating compounds and compounds capable ofremoving trace and heavy metals from liquids as well as water-solubleindustrial pollutants, such as PCBs, for example. p-GlcNAc and/orp-GlcNAc derivatives may be used in photographic applications. Forexample, the ability of p-GlcNAc and/or p-GlcNAc derivatives to chelatemetals, such as silver halides, may be utilized by contactingphotographic solutions to recast mats, such as thin membranes, ofp-GlcNAc and/or p-GlcNAc derivatives.

6. EXAMPLE Physical Characterization of Preparations of Pure p-GlcNAc

Presented in this Example, are circular dichroism (CD) and infra-redspectra (IR) analyses of p-GlcNAc and deacetylated p-GlcNAc membranes.

6.1. Materials and Methods

p-GlcNAC and commercial “chitin” preparations:

The p-GlcNAc used in the CD studies was prepared using the MechanicalForce purification method described, above, in Section 5.3.1.

Commercial “chitin” was purchased from NovaChem, Ltd., PO Box 1030Armdale, Halifax, Nova Scotia, Canada, B3L. 4K9.

The p-GlcNAC membranes used in the IR studies were prepared by eitherthe Mechanical Force purification method as described, above, in Section5.3.1, or by the Chemical/Biological purification method, as described,above, in Section 5.3.2, as indicated.

The commercial “p-GlcNAc” preparations were cast into membranes bydissolving in a dimethylacetamide solution containing 5% lithiumchloride, and layering onto distilled, deionized water until membranesprecipitated.

p-GlcNAC derivatives and treatments: The Deacetylated p-GlcNAC used inboth the CD and IR studies was prepared by treatment of the p-GlcNACwith 50% NaOH at 60° C. for 2 hours. The heat-denatured p-GlcNACmembranes used in the IR studies were modified by boiling in 0.2 mM EDTAfor 3 minutes. p-GlcNAc was autoclaved for 30 minutes at 122° C.

CD techniques: Solid state CD techniques were carried out essentiallyaccording to Domard (Domard, A., 1986, Int. J. Macromol. 8:243-246).

6.2. Results 6.2.1. CD Analysis

In the CD spectra obtained from untreated p-GlcNAc (FIG. 3A), theexpected n-π⁺ and π-π*⁺ optically active electronic transitions (220-185nM) were observed due to the presence of the carbonyl group in theacetyl moiety of p-GlcNAc. Such peaks are completely absent in the CDspectrum obtained from the deacetylated p-GlcNAc product, as shown inFIG. 3B.

6.2.2. IR Spectra Analysis

The IR spectra obtained in this study are consistent with the chemicalstructure of p-GlcNAc. Additionally, the sharp definition of each IRpeak is indicative of the presence of an ordered and regular (i.e.,pseudocrystalline) structure in the p-GlcNAc fibers. See FIG. 4A for theIR spectrum of p-GlcNAc purified via the Mechanical Force purificationmethod, and FIG. 4D for the IR spectrum of p-GlcNAc purified via theChemical/Biological method. For comparison, see FIG. 4B, whichdemonstrates the IR spectrum of a commercial “chitin” preparation.

The IR spectrum obtained from the autoclaved p-GlcNAc material (FIG. 4E)does not differ visibly from the IR spectrum observed in FIG. 4A. Thisdata indicates that the p-GlcNAc material may be sterilized byautoclaving with no loss of polymer structure.

7. EXAMPLE Purification of p-GlcNAC using the Mechanical ForcePurification Method

In this section, p-GlcNAC was purified using the Mechanical Forcetechnique described above, in Section 5.3.1.

7.1. Materials and Methods/Results

Diatom culture conditions: The diatom species Thalassiosira fluviatiliswas grown in culture according the procedures described, above, inSections 5.1 and 5.2.

SEM Procedures: The SEM techniques used here are as those described,below, in Section 12.1.

p-GlcNAc Purification procedure:p-GlcNAC was purified from the diatomculture by utilizing the Mechanical Force technique described above, inSection 5.3.1. Specifically, the p-GlcNAc fibers were separated from thediatom cell bodies by subjecting the contents of the culture to threeshort bursts of top speed mixing motion in a Waring blender. Total timeof the three bursts was about one second. The resulting suspension wascentrifuged at 3500 rpm in a Sorvall GS-4 fixed angle rotor, for 20minutes at about 10° C. The supernatant was decanted, and centrifugedagain, this time at 4000 rpm, in a Sorvall GS-4 fixed angle rotor for 20minutes at about 10° C. Once again, the supernatant was decanted andcentrifuged at 4000 rpm at 10° C. The final supernatant of the thirdcentrifugation was clear, with little, if any, visible flocs floating inthe liquid. The clear supernatant was decanted into a Buchner filtrationunit equipped with a Supor-800 polyether sulfone filter membrane with0.8 μm pore size (Gelman, Inc.), suction was then applied and the liquidwas filtered from the fiber suspension, allowing the fibers to becollected on the membrane. The collected fibers were washed with 1 literof distilled, deionized H₂O at 70° C. When almost all of the water hadbeen drained, fibers were washed, with suction, with 1 liter of 1 N HClat 70° C. When most of the acid solution had been drained, the fiberswere washed with 1 liter of distilled, deionized H₂O at 70° C., usingsuction. When most of the wash water had been drained, the fibers werewashed with 1 liter of 95% ethanol at room temperature, and vacuum wasapplied. The filter membrane on which the white fiber membrane had beencollected was then removed from the filtration unit and the membrane andits membrane support was dried in a drying oven at 58° C. for 20minutes, after which the membrane and its support was placed in adesiccator for 16 hours.

Following this purification procedure, the yield of p-GlcNAc from a 1000ml culture was 6.85 milligrams per liter of diatom culture. SEMphotographs of the membrane formed by the collection of the p-GlcNACfibers via this technique is shown in FIG. 6.

8. EXAMPLE Purification of p-GlcNAC Using the Biological/ChemicalPurification Method

In this section, p-GlcNAC was purified using two of theChemical/Biological techniques described above, in Section 5.3.2.Briefly, p-GlcNAC was purified via HF treatment, in one case, and viaacid treatment/neutralization in the second case.

8.1. Materials and Methods/Results

Diatom culture conditions: The diatom species Thalassiosira fluviatiliswas grown in a culture according to the procedures described, above, inSections 5.1 and 5.2.

SEM procedures: The techniques utilized in this study were as described,below, in Section 12.1.

Purification procedure: First, p-GlcNAC was purified by HF treatment,the results of which are shown in FIG. 7. Specifically, under a fumehood, 2.42 ml of a 49% (29N) HF solution was added to the diatomcontents of the culture, at room temperature, for each 1000 ml of thevolume of the original cell culture, resulting in a 0.07 M HF solution.The mixture was then shaken vigorously for about 30 seconds, causingpersistent foam to appear over the liquid. The container was allowed tostand undisturbed for 5-6 hours to allow heavy particulates to settle.At the end of this time, a layer of foam had formed, while the liquiditself was divided into two strata: first, a narrow, very dark greenlayer resting on the bottom of the container below a second, muchlighter colored grayish-green and murky phase which represented perhaps85-90% of the total volume of liquid. The foam layer was carefullysiphoned off, using a capillary glass tube and vacuum suction. Thegrayish cloudy supernatant was then siphoned off, with care being takennot to disturb the dark bottom layer, which consisted mainly of settledcell bodies, and was transferred to a separate plastic container. Thegrayish cloudy supernatant was allowed to stand undisturbed for anadditional 16 hours. The liquid was initially almost colorless, lightgrey, but not transparent. After 16 hours settling time, a small amountof foam remained on top of the main body of liquid and a small amount ofgreen matter had settled on the bottom of the container. The liquid waslighter in color, but still not transparent. The foam on top of theliquid was siphoned off as before. The main body of liquid was thencarefully siphoned off, leaving behind the small amount of settled greenmaterial at the bottom of the container. The liquid which had thus beenisolated, contained the majority of the p-GlcNAc fibers and someimpurities.

To remove proteins and other unwanted matter liberated by the diatomsduring the preceding steps in the procedure from the fiber-containingliquid, the suspension of fibers and cell remnants was washed withsodium dodecyl sulfate (SDS). Specifically, the necessary volume of a20% SDS solution was added to make the final concentration of the liquid0.5% SDS by volume. The container holding the liquid was sealed, securedin a horizontal position on a shaking machine, and agitated for 24 hoursat about 100 shakes a minute. Soon after shaking began, large flocs ofwhite p-GlcNAc fibers appeared in the suspension, and a considerableamount of foam accumulated in the head space of the containers. At theend of the SDS washing, the contents of the containers were transferredto a Buchner filtration equipment provided with a Supor-800 polyethersulfone filter membrane, with 0.8 micron pore size (Gelman, Inc.). Theliquid was filtered with suction, and the p-GlcNAc fibers in the liquidwere collected on the filter membrane.

The p-GlcNAc fibers collected on the filter membrane were then washedfurther. First, the fibers were washed with hot (70° C.) distilled,deionized H₂O, using three times the volume of the original suspension.With a water jet using distilled, deionized H₂O, the white fiber clumpscollected on the filter membrane of the Buchner filter were transferredto a Waring blender, and the fiber clumps were disintegrated with about10 short mixing bursts. The suspension of disintegrated fibers wastransferred to a Buchner filter funnel equipped with a polyether sulfonefilter membrane as described above, and the liquid was removed undersuction. The collected fibers were washed with 1000 ml of hot (70° C.)1N HCl solution, and subsequently further washed with 1000 ml hot (70°C.) distilled, deionized H₂O. Finally, the fibers were washed with 1000ml 95% ethanol at room temperature, and filtered to dryness. The fibermembrane and the filter membrane supporting the fiber membrane were thendried in a drying oven at 58° C. for 20 minutes. The membrane andmembrane support was then placed in a desiccator for 16 hours. Themembrane was then carefully detached from the filter membrane.

Second, p-GlcNAc was purified by using the acid treatment/neutralizationmethod described, above, in Section 5.3.2. Specifically, the p-GlcNAcwas processed as described earlier in this Section, until prior to theSDS wash step, at which point the solution was neutralized to a pH ofapproximately 7.0 by the addition of a 2.9M Tris solution. The p-GlcNAcyield from this particular purification procedure was 20.20 milligramsper liter of diatom culture, although, on average, approximately 60milligrams per liter diatom culture are obtained. SEM micrographs ofmembranes formed during the purification procedure are shown in FIGS. 8and 9A-9E.

9. EXAMPLE p-GlcNAc Deacetylation

A p-GlcNAc membrane was suspended in an aqueous 50% NaOH solution. Thesuspension was heated at 80° C. for 2 hours. The resulting deacetylatedmembrane was dried and studied by scanning electron microscopy, as shownin FIG. 11.

10. EXAMPLE p-GlcNAc Biocompatibility

In this Example, it is demonstrated that the p-GlcNAc of the inventionexhibits no detectable biological reactivity, as assayed by elutiontests, intramuscular implantation in rabbits, intracutaneous injectionin rabbits, and systemic injections in mice.

10.1. Materials and Methods 10.1.1. Elution Test

Conditions for the elution test conformed to the specifications setforth in the U.S. Pharmacopeia XXII, 1990, pp. 1415-1497 and to U.S.Pharmacopeia XXII, Supplement 5, 1991, pp. 2702-2703.

Cell culture: Mouse fibroblast L929 cell line (American Type CultureCollection Rockville, Md.; ATCC No. CCL1; NCTC clone 929) was utilized.A 24 hour confluent monolayer of L929 cells was propagated in completeMinimum Essential Medium (MEM).

p-GlcNAc: a solid membrane of p-GlcNAc which had been prepared accordingto the Mechanical Force method of purification described, above, inSection 5.3.1, was extracted in 20 ml serum-supplemented MEM as per U.S.Pharmacopeia XXII (1990) requirements.

Controls: Natural rubber was used as a positive control, and siliconewas used as a negative control. Controls were tested in the same manneras the test article, p-GlcNAc.

Extracts: Extracts were prepared at 37° C., in a humidified atmospherecontaining 5% carbon dioxide, for 24 hours. Extracts were evaluated fora change in pH, and adjustments were made to bring the pH to within+/−0.2 pH units of the original medium. Adjustments were made with HClto lower the extract pH or with NaHCO₃ to raise the extract pH. Extractswere sterile filtered by passage through a 0.22 micron filter, prior tobeing applied to the cell monolayer.

Dosing: 3 mls of p-GlcNAc or control extracts were used to replace themaintenance medium of cell cultures. All extracts were tested induplicate.

Evaluation Criteria: Response of the cell monolayer was evaluated eithervisually or under a microscope. The biological reactivity, i.e.,cellular degeneration and/or malformation, was rated on a scale of 0 to4, as shown below. The test system is suitable if no signs of cellularreactivity (Grade 0) are noted for the negative control article, and thepositive control article shows a greater than mild reactivity (Grade 2).The test article (i.e., p-GlcNAc) meets the biocompatibility test ifnone of the cultures treated with the test article show a greater thanmild reactivity. Grade Reactivity Description of Reactivity Zone 0 NoneDiscrete intracytoplasmic granules; No cell lysis 1 Slightly Not morethan 20% of the cells are round, loosely attached, and withoutintra-cytoplasmic granules; occasional lysed cells are present 2 MildNot more than 50% of the cells are round and devoid of intracytoplasmicgranules; extensive cell lysis and empty areas between cells 3 ModerateNot more than 70% of the cell layers contain rounded cells and/or arelysed 4 Severe Nearly complete destruction of the cell layers

10.1.2. Intramuscular Implantations

Animals: Healthy, New Zealand White Rabbits, male and female, (EasternRabbit Breeding Laboratory, Taunton, Mass.) were used. Rabbits wereindividually housed using suspended stainless steel cages. Upon receipt,animals were placed in quarantine for 8 days, under the same conditions,as for the actual test. Hardwood chips (Sani-chips™, J. P. Murphy ForestProducts, Montvale, N.J.) were used as non-contact bedding under cages.The animal facility was maintained at a temperature of 68°+/−3° F., witha relative humidity at 30-70%, a minimum of 10-13 complete air exchangesper hour, and a 12-hour light/dark cycle using full spectrum fluorescentlights. Animals were supplied with commercial feed (Agway ProLab,Waverly, N.Y.) under controlled conditions and municipal tap water adlibitum. No known contaminants were present in the feed, bedding, orwater which would be expected to interfere with the test results.Animals selected for the study were chosen from a larger pool ofanimals. Rabbits were weighted to nearest 10 g and individuallyidentified by ear tattoo.

p-GlcNAc: The p-GlcNAc used was as described, above, in Section 10.1.1.

Implantation Test: Two rabbits were used for each implantation test. Onthe day of the test, the animal skin on both sides of the spinal columnwas clipped free of fur. Each animal was anesthetized to preventmuscular movement. Using sterile hypodermic needles and stylets, fourstrips of the test p-GlcNAc (1 mm×1 mm×10 mm) were implanted into theparavertebral muscle on one side of the spine of each of two rabbits(2.5 to 5 cm from the midline, parallel to the spinal column, and about2.5 cm from each other). In a similar fashion, two strips of the USPnegative control plastic RS (1 mm×1 mm×10 mm) were implanted in theopposite muscle of each animal. Animals were maintained for a period of7 days. At the end of the observation period, the animals were weighedand euthanized by an injectable barbiturate, Euthanasia-5 (VeterinaryLaboratories, Inc., Lenexa, Kans.). Sufficient time was allowed toelapse for the tissue to be cut without bleeding. The area of the tissuesurrounding the center portion of each implant strip was examinedmacroscopically using a magnifying lens. Hemorrhaging, necrosis,discolorations and infections were scored using the following scale:0=Normal, 1=Mild, 2=Moderate, and 3=Severe. Encapsulation, if present,was scored by first measuring the width of the capsule (i.e., thedistance from the periphery of the implant to the periphery of thecapsule) rounded to the nearest 0.1 mm. The encapsulation was scored asfollows: Capsule Width Score None 0 up to 0.5 mm 1 0.6-1.0 mm 2 1.1-2.0mm 3 Greater than 2.0 mm 4

The differences between the average scores for the p-GlcNAc and thepositive control article were calculated. The test is considerednegative if, in each rabbit, the difference between the average scoresfor each category of biological reaction for the p-GlcNAc and thepositive control plastic implant sites does not exceed 1.0; or, if thedifference between the mean scores for all categories of biologicalreaction for each p-GlcNAc article and the average score for allcategories for all the positive control plastic implant sites does notexceed 1.0, for not more than one of four p-GlcNAc strips.

10.1.3. Intracutaneous Injections

Animals: New Zealand white rabbits were used and maintained asdescribed, above, in Section 10.1.2.

p-GlcNAc: A solid membrane of p-GlcNAc which had been prepared accordingto the mechanical force method of purification described, above, inSection 5.3.1, was placed in an extraction flask, to which 20 ml of theappropriate medium were added. Extractions were performed by heating to70° C. for 24 hours. Following this procedure, extracts were cooled toroom temperature. Each extraction bottle was shaken vigorously prior toadministration.

Intracutaneous Test: On the day of the test, animals were clipped freeof fur on the dorsal side. A volume of 0.2 ml of each p-GlcNAc extractwas injected intracutaneously at five sites on one side of each of tworabbits. More than one p-GlcNAc extract was used per rabbit. At fivesites on the other side of each rabbit, 0.2 ml of the correspondingcontrol was injected. Injection sites were observed for signs oferythema, edema, and necrosis at 24, 48, and 72 hours after injection.Observations were scored according to the Draize Scale for the ScoringSkin Reaction (USP Pharmacopeia XXII, 1990, 1497-1500; USP PharmacopeiaXXII, Supplement 5, 1991, 2703-2705) as shown in Table II, below: TABLEII Draize Scale for Scoring Skin Reactions Value Erythema and EscharFormation No erythema 0 Very slight erythema (barely perceptible) 1 Welldefined erythema 2 Moderate to severe erythema 3 Severe erythema (beetredness) to slight eschar 4 formation (injuries in depth) Total possibleerythema score = 4 Edema Formation No edema 0 Very slight edema (barelyperceptible) 1 Slight edema (edges are well defined by definite 2raising) Moderate edema (raised approximately 1 mm and 3 extendingbeyond area of exposure) Severe edema (raised more than 1 mm andextending 4 beyond area of exposure) Total possible edema score = 4All erythema and edema scores at 24, 48, and 72 hours were totaledseparately and divided by 12 (i.e., 2 animals×3 scoring periods×2scoring categories) to determine the overall mean score for the p-GlcNAcversus the corresponding control. Animals were weighed at the end of theobservation period and euthanized by injection of a barbiturate,Euthanasia-5 (Veterinary Laboratories, Inc., Lenexa, Kans.). The resultsof the test are met if the difference between the p-GlcNAc and thecontrol means reaction scores (erythema/edema) is 1.0 or less).

10.1.4. Systemic Injections

Animals: Albino Swiss mice (Mus musculus), female, (Charles RiverBreeding Laboratories, Wilmington, Mass.) were used. Groups of 5 micewere housed in polypropylene cages fitted with stainless steel lids.Hardwood chips (Sani-chips™, J. P. Murphy Forest Products, Montvale,N.J.) were used as contact bedding in the cages. The animal facility wasmaintained as a limited access area. The animal rooms were kept at atemperature of 68+/−3° F., with a relative humidity of 30-70%, a minimumof 10-13 complete air exchanges per hour, and a 12 hour light/dark cycleusing full spectrum fluorescent lights. Mice were supplied withcommercial feed and municipal tap water ad libitum. There were no knowncontaminants present in the feed, bedding, or water which would beexpected to interfere with the test results. Animals selected for thestudy were chosen from a larger pool of animals. Mice were weighed tothe nearest 0.1 g and individually identified by ear punch.

p-GlcNAc: The samples used were as described, above, in Section 10.1.1.Extracts were prepared according to the procedures described in Section10.1.3, above.

Systemic Injection Test: Groups of 5 mice were injected with eitherp-GlcNAc extract or a corresponding control article, in the same amountsand by the same routes as set forth below: Test Article or ControlArticle Injection Extracts Dosing Route Dose/Kg Rate 0.9% SodiumIntravenous 50 ml 0.1 ml/sec Chloride Injection, USP (0.9% NaCl) 1 in 20Alcohol Intravenous 50 ml 0.1 ml/sec in 0.9% Sodium Chloride InjectionUSP (EtOH:NaCl) Polyethylene Intraperitoneal 10 g — Glycol 400 (PEG 400)Cottonseed Oil Intraperitoneal 50 ml — (CSO)Extracts of the p-GlcNAc prepared with PEG 400, and the correspondingcontrol, were diluted with 0.9% NaCl, to obtain 200 mg of PEG 400 perml.For the Intracutaneous Test, PEG 400 was diluted with 0.9% NaCl toobtain 120 mg of PEG 400 per ml.

The animals were observed immediately after injection, at 24 hours, 48hours, and 72 hours after injection. Animals were weighed at the end ofthe observation period and euthanized by exposure to carbon dioxide gas.The requirements of the test are met if none of the animals treated withthe p-GlcNAc shows a significantly greater biological reactivity thanthe animals treated with the control article.

10.2. Results 10.2.1. Elution Test

The response of the cell monolayer to the p-GlcNAc test article wasevaluated visually and under a microscope. No cytochemical stains wereused in the evaluation. No signs of cellular biological reactivity(Grade 0) were observed by 48 hours post-exposure to the negativecontrol article or to the p-GlcNAc. Severe reactivity (Grade 4) wasnoted for the positive control article, as shown below in Table III:TABLE III REACTIVITY GRADES Control Articles p-GlcNAc Negative PositiveTime A B A B A B  0 Hours 0 0 0 0 0 0 24 Hours 0 0 0 0 4 4 48 Hours 0 00 0 4 4The p-GlcNAc of the invention, therefore, passes requirements of theelution test for biocompatibility, and, thus, is non-cytotoxic.

10.2.2. Intramuscular Implantations

Both rabbits (A and B) tested increased in body weight and exhibited nosigns of toxicity. See Table IV for data. In addition, there were noovert signs of toxicity noted in either animal. Macroscopic evaluationof the test and control article implant sites showed no inflammation,encapsulation, hemorrhage, necrosis, or discoloration. See Table IV forresults. The test, therefore, demonstrates that the p-GlcNAc assayedexhibits no biological reactivities, in that, in each rabbit, thedifference between the average scores for all of the categories ofbiological reaction for all of the p-GlcNAc implant sites and theaverage score for all categories for all the control implant sites didnot exceed 1.0. TABLE IV IMPLANTATION TEST (Macroscopic Observations)Test Article: p-GlcNAc Animal Species: Rabbit TISSUE SITE: TEST CONTROLT1 T2 T3 T4 AVERAGE C1 C2 AVERAGE Animal #: A Inflammation 0 0 0 0 0 0 00 Encapsulation 0 0 0 0 0 0 0 0 Hemorrhage 0 0 0 0 0 0 0 0 Necrosis 0 00 0 0 0 0 0 Discoloration 0 0 0 0 0 0 0 0 Total 0 0 0 0 0 0 MEAN SCORE:0 0 0 0 0 0 (total/5) AVERAGE CONTROL VALUE: 0 Animal #: B Inflammation0 0 0 0 0 0 0 0 Encapsulation 0 0 0 0 0 0 0 0 Hemorrhage 0 0 0 0 0 0 0 0Necrosis 0 0 0 0 0 0 0 0 Discoloration 0 0 0 0 0 0 0 0 Total 0 0 0 0 0 0MEAN SCORE: 0 0 0 0 0 0 (total/5) AVERAGE CONTROL VALUE: 0

10.2.3. Intracutaneous Test

All of the animals increased in weight. See Table V for data. There wereno signs of erythema or edema observed at any of the p-GlcNAc or controlarticle sites. Overt signs of toxicity were not observed in any animal.Because the difference between the p-GlcNAc and control article meanreaction scores (erythema/edema) was less than 1.0, the p-GlcNAc meetsthe requirements of the intracutaneous test. See Table VI for results.Therefore, as assayed by this test, the p-GlcNAc demonstrates nobiological reactivity. TABLE V Intracutaneous and Implant Tests BodyWeights and Clinical Observations Test Article: p-GlcNAc Animal Species:Rabbit Body Weight Animal (Kg) Weight Signs of Group # Sex Day 0 ChangeToxicity* Day 3 0.9% NaCl 23113 Male 2.51 2.55 0.04 None & CSO 0.9% NaCl23114 Female 2.43 2.46 0.03 None & CSO EtOH: NaCl 23115 Male 2.47 2.500.03 None & PEG 400 EtOH: NaCl 23116 Female 2.59 2.63 0.04 None & PEG400 Day 7 Implant A Male 2.74 2.80 0.06 None B Female 2.66 2.74 0.08None*Summary of Observations Day 0 Through Day 7 (Implant) and Day 0 throughDay 3 (Intracutaneous).

TABLE VI INTRACUTANEOUS TEST DRAIZE SCORES Test Article: p-GlcNAc (T =test, C = control) Animal Species: Rabbit Animal SITE NUMBERS SCORING(ER/ED) Averages ID # Vehicle T-1 C-1 T-2 C-2 T-3 C-3 T-4 C-4 T-5 C-5Time: T C NaCl Extract 23113 NaCl 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 24 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 48 hr. 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 72 hr. 0/0 0/0 Total 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 23114 NaCl 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 24 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 48 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 72 hr. 0/00/0 Total 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 CSO Extract23113 CSO 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 24 hr. 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 48 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 72 hr. 0/0 0/0 Total 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 23114 CSO 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 24 hr. 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 48 hr. 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 72 hr. 0/0 0/0 Total 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 NaCl/EtOH Extract 23115 NaCl 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 24 hr. 0/0 0/0 EtOH 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 48 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 72 hr.0/0 0/0 Total 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 23116 NaCl0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 24 hr. 0/0 0/0 EtOH 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 48 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 72 hr. 0/0 0/0 Total 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 PEG Extract 23115 PEG 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 24hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 48 hr. 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 72 hr. 0/0 0/0 Total 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 23116 PEG 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 24 hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 48hr. 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 72 hr. 0/0 0/0 Total0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

10.2.4. Systemic Test

All of the mice treated with the p-GlcNAc extract or the control articleincreased in weight. See Table VII for data. In addition, there were noovert signs of toxicity observed in any p-GlcNAc or control animal. SeeTable VI for results. It is concluded, therefore, that none of thep-GlcNAc test animals showed a significantly greater biologicalreactivity than the animals treated with the control article. TABLE VIIANIMAL WEIGHTS AND CLINICAL OBSERVATIONS Dose Body Weight (g) Signs ofGroup Sex (ml) Animal # Day 0 Day 3 Weight Change Toxicity* NaCl: Female1.03 I. 20.6 22.8 2.2 None EtOH Female 1.06 II. 21.1 23.4 2.3 None TestFemale 1.02 III. 20.4 22.6 2.2 None 50 ml/kg Female 1.11 IV. 22.2 24.52.3 None Female 1.05 V. 21.0 23.2 2.2 None Mean 21.1 23.3 SD+/− 0.7 0.7NaCl: Female 1.04 VI. 20.7 23.2 2.5 None EtOH Female 1.04 VII. 20.8 23.52.7 None Control Female 1.02 VIII. 20.3 22.3 2.0 None 50 ml/kg Female0.91 IX. 18.2 20.6 2.4 None Female 0.94 X. 18.7 20.9 2.2 None Mean 19.722.1 SD+/− 1.2 1.3 PEG Female 1.02 XI. 20.3 22.7 2.4 None Test Female0.96 XII. 19.2 21.4 2.2 None 10 ml/kg Female 0.95 XIII. 18.9 21.6 2.7None Female 1.05 XIV. 20.9 22.7 1.8 None Female 0.94 XV. 18.7 21.2 2.5None Mean 19.6 21.9 SD+/− 1.0 0.7 PEG Female 1.01 XVI. 20.1 22.3 2.2None Control Female 0.99 XVII. 19.8 22.0 2.2 None 10 g/kg Female 1.10XVIII. 22.0 24.3 2.3 None Female 1.07 XIX 21.4 23.6 2.2 None Female 1.03XX. 20.6 22.4 1.8 None Mean 20.8 22.9 SD+/− 0.9 1.0*Summary of observations - 0, 4, 24, 48, and 72 h after injection

11. EXAMPLE p-GlcNAc Reformulation

In the Working Example presented in this Section, a p-GlcNAc membrane(16.2 mg) was dissolved in 1 ml of a dimethylacetamide solutioncontaining 5% LiCl. The p-GlcNAc-containing solution was placed in asyringe and extruded into 50 ml of pure water to precipitate a fiber.The resulting fiber was studied with scanning electron microscopy, asshown in FIG. 10.

12. EXAMPLE Cell Attachment to p-GlcNAc

In this working example, it is demonstrated that p-GlcNAc represents anefficient substrate for cell attachment and growth in culture.

12.1. Materials and Methods

Cells: The transformed mouse 3T3 fibroblast cell line was used, and wasgrown in DMEM supplemented with 10 fetal bovine serum (FBS).

p-GlcNAc membranes: p-GlcNAc was prepared according to the methodsdescribed, above, in Sections 5.3.1 and 8.

p-GlcNAc membranes were initially stuck to a #1 (18 mm) Corning coverglass using one drop of water, and were attached by autoclaving at 121°C. for 30 minutes. Membranes prepared in this manner were then placed inculture wells of 6 well culture plates.

Cell counts: Cell numbers were determined in media by direct countingwith a hemocytometer, and on matrix by first rinsing membranes withfresh medium DMEM+10% FBS) followed by treatment with trypsin (10%, at37° C. for 5 minutes) prior to counting.

SEM operating conditions: A Zeiss 962 instrument was utilized with anaccelerating voltage of 10 kv, and a working distance of 15 mm. Polaroidtype 55 p/n (u4) was utilized at various magnifications, as indicated.Sample coat:carbon coat (100 å) & 100 å aupd.

Specimen preparation: For primary fixation, the culture growth mediumwas replaced with 2% glutaraldehyde in Eagle's DMEM without serum.Several changes were performed to ensure a complete transition fromgrowth media to fixative. Fixation proceeded for 0.5 hours at roomtemperature. Cover slips were transferred to fresh vials containing 2%Glutaraldehyde in 0.1M Na Cacodylate at pH 7.2 with 0.1M Sucrose andfixed for a further 1.5 hours at room temperature.

Dehydration, CPD, Mount and Sputter Coating:

Samples were rinsed in 0.1M Na Cacodylate pH 7.2, and cover slips weretransferred to a CPD holder. Dehydration was performed in ethanol series(30%, 50%, 75%, 85%, 95% and 3×100%, 5 mins each), and samples werecritical point dried. Cover slips were then mounted on Al stubs, carboncoated, using vacuum evaporator (@ 100 Å) and sputter coated with 100 ÅAuPd.

12.2. Results

p-GlcNAc membranes were tested for an ability to form a substrate onwhich cells may be grown in culture. Mouse fibroblast cells were grownin wells in the presence or absence of p-GlCNAc membranes and cellcounts were taken daily to assay the viability of cultures. The resultsof one such series of cell counts is shown in FIG. 14. As indicated, byday 5 after plating, only the wells containing p-GlcNAc membranes wereable to continue to sustain viable cells, demonstrating that p-GlcNAcmembranes are capable of acting as efficient substrates for thecontinued growth of cells in culture.

Further, the SEM micrographs depicted in FIG. 15 show healthy cellsstrongly attached to p-GlcNAc membranes.

13. EXAMPLE p-GlcNAc/Collagen Hybrids

Presented in this Working Example is the formation and characterizationof a p-GlcNAc/collagen hybrid material.

13.1. Materials and Methods

Materials: Bovine Type I collagen was used in preparation of the hybridsdescribed in this study. p-GlcNAc was prepared according to themechanical force method described, above, in Section 5.3.2.

Hybrid Preparation: Collagen (10 milligrams/ml) and p-GlcNAc (0.25milligrams/ml) aqueous suspensions were mixed, in different ratios,frozen in liquid N₂ (−80° C.), held at −9° C. for 4 hours, andlyophilized. Material was dehydrothermally cross-linked under vacuum(approximately 0.030 Torr) at 60° C. for 3 days.

Cell Culture: Mouse 3T3 fibroblast cells were grown on thecollagen/p-GlcNAc hybrids produced. Standard culturing procedures werefollowed, and SEM micrographs were taken after 8 days in culture.

13.2. Results

Collagen and p-GlcNAc aqueous suspensions were mixed in differing ratios(namely, 3:1, 1:1, 2:2, and 1:3 collagen:p-GlcNAc suspension ratios),frozen, lyophilized, and crosslinked. Such a procedure yieldedcollagen/p-GlcNAc slabs. SEM micrographs of the resulting materials areshown in FIG. 16 B-E. FIG. 16A represents a collagen-only controlmaterial. Note the porous structure of the hybrid material.

The collagen/p-GlcNAc hybrids of the invention provide an efficientthree-dimensional structure for the attachment and growth of cells, asshown in the SEM micrographs in FIGS. 17A-D.

14. EXAMPLE NMR Characterization of Pure Preparations of p-GlcNAc

Presented in this Example is an NMR (nuclear magnetic resonance)analysis of a pure p-GlcNAc preparation.

14.1. Materials and Methods

p-GlcNAc preparations: The p-GlcNAc used in the NMR studies describedhere was prepared using the chemical purification method described,above, in Section 5.3.2, with hydrofluoric acid utilized as the chemicalreagent.

NMR techniques: Solid state NMR data was obtained using a Bruker 500 MHNMR spectrometer. Computer image analysis was used to transform the rawNMR spectrum data so as to eliminate background and to normalizebaselines. An example of such transformed data are shown in FIG. 18.Transformed NMR curves such as that in FIG. 18 were used to obtain areasfor every carbon atom type, and then to calculate the ratios of CH3(area) to C-atom (area). Such values, obtained as described, areprovided in FIG. 20.

14.2. Results

Solid state NMR data was obtained by measuring the C¹³-NLR spectrum of a500 mg sample of p-GlcNAc. A typical NMR spectrum is shown in FIG. 19.The individual peaks represent the contribution to the spectrum of eachunique carbon atom in the molecule. The relative percentage of each typeof carbon atom in the molecule was determined dividing the area of thepeak generated by that carbon species by the total sum of the areasunder all of the NMR peaks obtained in the spectrum. Thus, it waspossible to calculate the ratio of each of the atoms of the moleculemeasured by a reference atom. All p-GlcNAc molecules consist ofN-acetylated glucosamine residues having C1, C2, C3, C4, C5 and C6atoms, by definition. The ratio, then, of the area of the N-acetyl CH3carbon atom peak to the areas of any of the glucosamine residue carbonatom peaks, above, should be 1.0 if all of the glucosamine residues inthe polymer are N-acetylated. Data such as those in FIG. 20 were used toobtain values for the CH3 (area) ratios.

The calculated ratios in FIG. 20 are in many cases equal to or nearlyequal to 1.0, within experimental error, e.g. CH3/C2=1.097,CH3/C6=0.984, CH3/C5=1.007, CH3/C1=0.886. These results are consistentwith the conclusion that the p-GlcNAc material of the invention is freeof contaminants and is fully acetylated (i.e. that essentially 100% ofthe glucosamine residues are N-acetylated).

15. EXAMPLE Synthesis and Biological Characterization of Controlled PoreSize Three-Dimensional p-GlcNAc Matrices

Described below, are methods for the production of three-dimensionalp-GlcNAc based porous matrices having controlled average pore sizes.Such matrices have a variety of important applications, particularly,for example, as means for the encapsulation of cells. Such cellencapsulation compositions are useful as transplantable cell-basedtherapeutics, and in other cell & tissue engineering applications suchas in cartilage regeneration. The capability to manipulate themorphology and dimensionality of p-GlcNAc materials, as demonstratedhere, provides a powerful tool in expanding the potential applicationsof the p-GlcNAc material of the invention.

15.1. Materials and Methods

p-GlcNAc starting material: p-GlcNAc was prepared using the chemicalpurification method described, above, in Section 5.3.2, withhydrofluoric acid utilized as the chemical reagent.

Matrix formation: Suspensions (5 mls) containing 20 mg p-GlcNAc sampleswere made in the solvents listed below in Section 15.2, prior tolyophilization. Samples were then poured into wells of tissue culturedishes and frozen at −20° C. The frozen samples were then lyophilized todryness, and the resulting three-dimensional matrices were removed.

Scanning electron microscopy techniques: The procedures utilized herewere performed as described, above, in Section 12.1. The images shown inFIGS. 21A-G. are 200× magnifications of the matrix material, and a scalemarking of 200 microns is indicated on each of these figures.

15.2. Results

p-GlcNAc suspensions were obtained with each of the following solvents,as described, above, in Section 15.1:

A. Distilled water

B. 10% methanol in distilled water

C. 25% methanol in distilled water

D. Distilled water only

E. 10% ethanol in distilled water

F. 25% ethanol in distilled water

G. 40% ethanol in distilled water

Samples of matrix formed using each of the solvents were subjected toscanning electron microscopic (SEM) analysis, as shown in FIGS. 21A-G.These figures reveal that the average matrix pore size decreases as thepercentage of either methanol or ethanol increases in each suspension.

Specifically, pore diameter in the two water suspensions (FIGS. 21A and21D) approach 200 microns on average. Pore size in the samples depictedin FIGS. 21C and 21F (25% methanol and ethanol, respectively) arebetween 30 and 50 microns on average.

The results shown here suggest that while both ethanol and methanol maybe successfully used to control p-GlcNAc pore size, ethanol may be moreefficient than methanol.

16. EXAMPLE Cell Growth on Three-Dimensional Porous p-GlcNAc Matrices

Described in this Section are results demonstrating the successful useof three-dimensional p-GlcNAc porous matrices as substrates for theculturing of cells.

16.1. Materials and Methods

p-GlcNAc starting material: p-GlcNAc was prepared using the chemicalpurification method described, above, in Section 5.3.2, withhydrofluoric acid utilized as the chemical reagent.

Matrix formation: Three-dimensional p-GlcNAc matrices were prepared bythe lyophilization of suspensions of p-GlcNAc in water, water-ethanol,or water-methanol mixtures.

Suspensions (5 mls) containing 20 mgs p-GlcNAc were prepared in thefollowing solvents prior to lyophilization:

1. Distilled water only

2. 10% methanol in distilled water

3.25% methanol in distilled water

4. Distilled water only

5. 10% ethanol in distilled water

6. 25% ethanol in distilled water

7.40% ethanol in distilled water

Samples were poured into circular wells of plastic tissue culture dishesand were frozen at −20° C. The frozen samples were then lyophilized todryness, and the resulting three-dimensional matrices were removed.Samples of each matrix were subjected to scanning electron microscopic(SEM) analysis.

Cells: Mouse embryo BALBC/3T3 fibroblast cell line (clone A31), obtainedfrom the ATCC, were used for culturing on the three-dimensional porousp-GlcNAc matrices.

Culturing conditions: One cm² samples of porous matrices were placed intissue culture wells and were covered with a standard tissue-culturegrowth medium. Each well was seeded and cells were cultured for 6 daysat 37° C. in a CO₂ incubator (5% CO₂).

SEM procedures: Matrix samples were fixed and subjected to SEM analysisas described, above, in Section 12.1. The matrices were prepared bylyophilizing p-GlcNAc in distilled water. Images vary in magnificationfrom 100× to 5000×, as indicated in figure legends (FIGS. 22A-G).

16.2. Results

SEM photographs of p-GlcNAc matrices containing attached mousefibroblast cells attached are shown in FIGS. 22A-G. These photographsshow that the three-dimensional p-GlcNAc matrices contain attached mousefibroblast cells. Further, the photographs reveal that there is a closeinteraction and connection between the cells and the p-GlcNAc matrixmaterial. It is also notable that the cells have a roundedthree-dimensional morphology which is different from the flat, spreadshape of the cells when cultured directly onto plastic culture dishes.Cell viabilities were determined to be greater than 95%.

17. EXAMPLE p-GlcNAc Successfully Reduces and Prevents Post-SurgicalAdhesions

The Example presented herein demonstrates the successful use of p-GlcNAcmaterials, specifically a p-GlcNAc membrane and gel formulation, toreduce or prevent the formation of post-surgical adhesions in a seriesof animal models for such adhesions.

17.1. Materials and Methods

Synthesis p-GlcNAc-lactate: p-GlcNAc membrane starting material wasproduced by the chemical method, as described, above, in Section 5.3.2,with hydrofluoric acid utilized as the chemical reagent.

The p-GlcNAc was converted to deacetylated p-GlcNAc by the followingmethod. (It should be noted that approximately 1.4 g of p-GlcNAc areneeded to produce 1 g of p-GlcNAc lactate, given the expected loss inmass of approximately 15% which occurs upon deacetylation). In astoppered flask approximately 200 mg of p-GlcNAc membrane material weremixed vigorously with approximately 200 ml 60% NaOH. The vigorousshaking served to separate the p-GlcNAc membrane material to thegreatest extent possible. The NaOH solution used was made at least 12hours before use. The reaction flasks were placed in an 80° C. waterbath for 6 hrs, with periodic shaking to separate and wet the p-GlcNAcmaterial. After 6 hrs, the samples were taken from the water bath andthe NaOH solution was immediately decanted. The membrane materials werewashed with ddH₂O, at room temperature, until a pH of 7 was reached. Themembranes were removed from the water and dried on a Teflon sheet.

At this point a 2 mg sample was collected for C, H, N analysis in orderto determine the extent of deacetylation. Further, solubility of thedeacetylated material in 1% acetic acid was checked, with a solubilityof 1 mg/ml indicating that the p-GlcNAc material was appropriatelydeacetylated.

The partially deacetylated pGlcNAc was then converted to pGlcNAc-lactateusing the following method: Sufficient 2-propanol (containing 10% water)to wet all of the partially deacetylated pGlcNAc material and to allowfor stirring was added to 1 g of the partially deacetylated p-GlcNAc ina 250 ml Erlenmeyer flask. (Approximately 30 mls 2-propanol arerequired.) 2-propanol must be reagent grade, and fresh prior to eachsynthesis. With stirring, 1.1 mL of a 50% aqueous lactic acid solutionis added. Lactic acid should be reagent grade, and must be analyzed todetermine the exact concentration of available (i.e., non-esterified)lactic acid present. This was generally accomplished by titration with0.1N NaOH to the phenolphthalein end-point (pH 7.0). The concentrationof lactic acid used must be constant, i.e., must be +/−1 percent, foreach p-GlcNAc synthesis. The mixture was allowed to stir for at leasttwo hours at room temperature. It is possible to add low heat in orderto increase the reaction rate. Reaction time may be extended, or theamount of 50% aqueous lactic acid may be increased to ensure that thereaction goes to completion. The contents of the flasks were finelyfiltered through a Buchner funnel using quantitative ashless filterpaper. The material was washed with 15 ml of anhydrous 2-propanol. Thematerial was allowed to air dry in a fume hood for 2 hours and thenplaced in an oven at 40° C. overnight. For every gram of partiallydeacetylated p-GlcNAc starting material, a final p-GlcNAc-lactate weightof approximately 1.4 g, (i.e., an increase of 40% in mass) should beobtained.

Formulation of p-GlcNAc-lactate as a gel: The p-GlcNAc-lactate materialwas formulated into a gel as follows: p-GlcNAc-lactate material wasdissolved in distilled and deionized water to a concentration of between0.1-4.0% p-GlcNAc-lactate, by weight. Reagent grade propylene glycol(2-propanediol) was then added to a final propylene glycol concentrationof between 1-10%. In some cases, a preservative was added to preventbacterial and/or fungal contamination of the product. Typically,concentrations of p-GlcNAc-lactate of between 0.1%-4.0% were prepared.The viscosity of these preparations increases dramatically as thep-GlcNAc-lactate percentage increases, such that formulations having0.5% or more of the p-GlcNAc-lactate behave as gels.

Animal Models:

Sprague-Dawley rats: Adhesions are produced in this model by abrading orirritating the serosal surface of the cecum and apposing it to an areaof parietal peritoneum. The success rate for inducing adhesions incontrol animals with this method is reported to be at an average 80%.

Specifically, the surgical procedure for inducing post-surgicaladhesions in these rats involved the following. Animals were placed indorsal recumbency and prepared and draped accordingly for asepticsurgery. Abdominal cavities were exposed through a midline incision. Anarea, approximately 0.5 cm×1.0 cm, of parietal peritoneum on the leftabdominal wall was carefully excised, removing a thin layer of muscle,along with the peritoneum.

The cecum was then elevated and isolated. An area, approximately 0.5cm×1.0 cm, on the lateral surface of the proximal end of the cecum wasabraded by rubbing ten times with a dry gauze. The cecum was thenscraped with a scalpel blade to cause minute petechial hemorrhages. Thececal abrasion and the peritoneal incision were left exposed for 15minutes.

After 15 minutes, the test article (i.e., the p-GlcNAc material) orcontrol article was applied to the cecal wound. The cecal abrasion andthe peritoneal wound were then apposed and held in contact with Allistissue forceps for an additional 15 minutes.

The cecum was then replaced into the abdomen such that the abraded areaof the cecum was adjacent to the peritoneal site. The abdominal incisionwas closed and the animal was allowed to recover from the anesthesia.

Fourteen days after surgery, animals were euthanized and the abradedarea was examined for the formation of post-surgical adhesions. Ifadhesions were present, the entire area involved in the adhesion (i.e.,body wall, test or control article, and internal organs) were dissectedfree of the animal.

The extent of involvement and tenacity of adhesions were evaluatedaccording to the following scales: Extent of involvement scores: 0 noadhesion 1 adhesion <= 25% of the area 2 adhesion <= 50% of the area 3adhesion <= 75% of the area 4 adhesion > 75% of the area TenacityScores: 0 no adhesion 1 adhesion freed with blunt dissection 2 adhesionfreed with aggressive dissection 3 adhesion requiring sharp dissection

Additional animal models: Pig and horse large animal bowel models wereused to assess the prevention of peritoneal adhesions.

Surgical procedure: The animals were placed in dorsal recumbency andprepared and draped accordingly for aseptic surgery. The abdominalcavity was exposed through a midline incision. The small intestine waselevated and a 2 cm×2 cm section was identified, extensively abraded(approximately 200 strokes using a scalpel), and allowed to dry for 10minutes. The test article (i.e., p-GlcNAc material) or control articlewas then applied to the abraded wound, and the wounded section of thesmall intestine was replaced into the abdomen. In such a large boweltype of animal model, six wounds, each separated by 4 inches of bowelfrom the adjacent wound provides an environment highly prone to formadhesions. Following induction of the last of the wounds, the abdominalincision is closed and the animal is allowed to recover from theanesthesia.

Analysis of peritoneal adhesions: Twenty one days after surgery, animalswere euthanized and the abraded area was examined, with adhesionformation being evaluated following a procedure similar to that of theSprague-Dawley rat cecum model.

17.2. Results

When injury occurs, the body sets in motion a complex set of responsesdesigned to restore the injured area. In the final stages of healing,connective tissue forms at the wound site to regenerate the bodystructure and protect the affected area from further damage. In someinstances this cascade of events does not work properly and can lead tolife threatening conditions.

For example, as a visceral organ heals following surgery, a fibrin clotgenerated during the surgical procedure may invade the surface ofadjoining organs forming a link which allows for fibroblast migration.This migration leads to collagen deposition and tissue growth, which inturn causes the organs involved to adhere to one another.

Such adhesions, referred to as post-surgical adhesions, may producepain, obstruction and malfunction by distorting the organ or organsinvolved. Immobilized joints, intestinal obstruction and infertility areoften linked to the formation of post-surgical adhesions. Furthermore,post-surgical adhesion will complicate and extend the length of futuresurgical procedures in the surrounding region. This last issue is ofparticular relevance to open heart surgeries and cesarean sectionobstetrical procedures where additional surgeries may be required. Theformation of adhesions is very common following abdominal,cardiovascular and orthopedic surgical procedures.

When adhesions become pathological and seriously interfere with organfunction, surgical adhesiolysis (sharp or blunt dissection of theadhesion in conjunction with meticulous surgical techniques) is thetreatment that is currently used to eliminate adhesions. In 1991,approximately 500,000 adhesiolysis procedures were performed in the U.S.This procedure is, however, notoriously ineffective, with the frequencyof recurrence of adhesion formation reported to be as high as 90%.Further, no other technique or composition has proven effective in theprevention of such post-surgical adhesions.

The results presented herein, therefore, are significant in that theydemonstrate the effectiveness of the p-GlcNAc materials of the inventionfor the prevention of post-surgical adhesions. Specifically, the resultspresented here demonstrate the efficacy of p-GlcNAc based solid andliquid formulations as barriers to the formation of abdominalpost-surgical adhesions in accepted rat and pig animal model systems.

One of the accepted animal models used to study adhesion formationemploys visceral-parietal peritoneal adhesions in Sprague-Dawley rats.Both partially deacetylated p-GlcNAc membranes and p-GlcNAc-lactate gelformulations prevented and/or considerably reduced the incidence ofadhesion formation as compared with either non-treated controls ortreated with InterCEED™ (Johnson & Johnson), the only commerciallyavailable product for this indication. Specifically, a total of 18 ratswere used to test p-GlcNAc-lactate gel formulations. 12 animals wereused as controls, with 6 receiving no treatment and 6 receivingInterCeed™. 6 animals received 0.25% p-GlcNAc-lactate gel, 10% propyleneglycol, water. Animals receiving the p-GlcNAc-lactate gel treatmentshowed a significantly reduced incidence of postoperative adhesionformation, compared to either of the controls, as shown, below, in TableVIII. TABLE VIII Extent of Involvement Tenacity Control (No treatment) 1+/− 2.1 1 +/− 1.5 InterCEED ™ 1 +/− 1.8 1 +/− 1.5 p-GlcNAc-lactate gel 0+/− 0.8 1 +/− 1.2

Partially deacetylated p-GlcNAc membranes were also tested for theirability to reduce or prevent the occurrence of post-surgical adhesionsin the rat animal model. A total of 22 rats were used in the study. 12animals were used as controls, with 6 receiving no treatment and 6receiving InterCEED™. Ten animals each received a 1 cm×1 cm membrane ofan approximately 60% deacetylated p-glcNAc formulation. The animalswhich received the partially deacetylated p-GlcNAc membrane showed asignificant reduction in the incidence of formation of postoperativeadhesions, as compared with the non-treated controls and InterCEED™, asshown, below, in Table IX. TABLE IX Extent of Involvement TenacityControl (No treatment) 3 +/− 1.8 1 +/− 0.6 InterCEED ™ 3 +/− 1.6 1 +/−0.4 p-GlcNAc-membrane 1 +/− 0.8 1 +/− 0.3

Large animal bowel models for the prevention of peritoneal adhesionswere also used to test p-GlcNAc compositions. Specifically, six pigs andone horse were used to study both the partially deacetylated p-GlcNAcmembrane and the p-GlcNAc-lactate gel. The partially deacetylatedp-GlcNAc membrane consisted of a 2 cm×2 cm piece of 60% deacetylatedp-GlcNAc membrane, while the p-GlcNAc-lactate gel consisted of 0.25%p-GlcNAc lactate formulated with 10% propylene glycol and water. Controlanimals received no treatment to the wounded site.

The results of these large animal studies revealed that, while thecontrol sites formed multiple adhesions and scar tissue in thesurrounding site, both the p-GlcNAc membrane and gel formulationseffectively reduced the formation of adhesions.

Samples from control and treated sites were additionally examined usingSEM, which showed an increased amount of fibrosis in the control sitesas compared to the treated tissues.

18. EXAMPLE Biodegradability of p-GlcNAc Materials

The Example presented in this Section demonstrates that p-GlcNAcmaterials of the invention may be prepared which exhibit controllable invitro and in vivo biodegradability and rates of resorption.

18.1. Materials and Methods

p-GlcNAc materials: Prototype I was made by the method described, above,in Section 5.3.2, via the chemical method, with hydrofluoric acid beingutilized as the chemical reagent. Prototype I represented 100%acetylated p-GlcNAc.

The p-GlcNAc starting material of prototype 3A was made by the methoddescribed, above, in Section 5.3.2, via the chemical method, withhydrofluoric acid being utilized as the chemical reagent. The p-GlcNAcmaterial was then deacetylated by the method described, above, inSection 5.4. Specifically, the p-GlcNAc material was treated with a 40%NaOH solution at 60° C. for 30 minutes. The resulting prototype 3A wasdetermined to be 30% deacetylated.

The p-GlcNAc starting material of prototype 4 was made by the methoddescribed, above, in Section 5.3.2, via the chemical method, withhydrofluoric acid being utilized as the chemical reagent. The p-GlcNAcmaterial was then deacetylated by treatment with a 40% NaOH solution at60° C. for 30 minutes. Next, the fibers were suspended in distilledwater, frozen at −20° C., and lyophilized to dryness. Prototype 4 wasalso determined to be 30% deacetylated.

Abdominal implantation model: Sprague Dawley albino rats were utilizedfor the abdominal implantation model studies. Animals were anesthetizedand prepared for surgery, and an incision was made in the skin andabdominal muscles. The cecum was located and lifted out. A 1 cm×1 cmmembrane of p-GlcNAc material was placed onto the cecum, and theincision was closed with nylon. Control animals were those in which nomaterial was placed onto the cecum.

Animals were opened at 14 and 21 days post implantation. Photographswere taken during the implant and explant procedures (FIGS. 23A-E).Samples of cecum were prepared for histopathology after the explantprocedure.

p-GlcNAc in vitro degradation lysozyme-chitinase assay: The assay is acalorimetric assay for N-acetyl glucosamine, and was performed asfollows: 150 μl of a reaction sample was pipetted into 13×100 mm glassdisposable test tubes, in duplicate. 25 μl of 0.25M potassium phosphatebuffer (pH 7.1) was added to each test tube, followed by the addition of35 μl of 0.8M potassium borate solution (pH 9.8). Tubes were immediatelyimmersed into an ice-bath for a minimum of 2 minutes. Samples were thenremoved from the ice-bath, 1 ml of freshly prepared DMAB reagent wasadded, and the samples were vortexed. DMAB (Dimethyl aminobenzaldehyde)reagent was made by adding 70 mls of glacial acetic acid and 10 mls of11.6N (concentrated) HCl to 8 grams of p-dimethyl aminobenzaldehyde.Samples were then incubated at 37° C. for 20 minutes.

To prepare a standard curve, the following procedure was utilized. AGlcNAc stock solution was diluted to 0.1 mg/ml with 0.010M sodiumacetate buffer (pH 4.5), and 0 μl, 20 μl, 30 μl, 90 μl or 120 μl of thediluted GlcNAc solution was added to a set of test tubes. This wasfollowed by the addition of 150 μl, 130 μl, 60 μl or 30 μl,respectively, of 0.010M sodium acetate buffer (pH 4.5) to the testtubes. Next, 25 μl of 0.25M potassium phosphate buffer (pH 7.1) and 35μl of 0.8M potassium borate buffer (pH 9.8) were added to each testtube. A duplicate set of test tubes is prepared by the same procedure.

The test tubes are capped and boiled at 100° C. for exactly 3 minutes.The tubes are then immersed in an ice bath. The tubes are removed fromthe ice bath and 1 ml of DMAB reagent, freshly prepared according to themethod described above in the Section, is added to each tube. The tubesare incubated at 37° C. for 20 minutes. The absorbance of the contentsof each tube is read at 585 nM. Absorbance should be read as quickly aspossible. The standard curve is plotted on graph paper and used todetermine the concentration of N-acetyl glucosamine in the reactionsamples. A typical standard curve is shown in FIG. 23.

18.2. Results

The in-vitro biodegradability of p-GlcNAc materials was studied inexperiments which assayed the relative susceptibility of p-GlcNAcmembrane materials to degradation by lysozyme. p-GlcNAc membranes wereexposed to an excess of lysozyme in a 10 mM acetate buffer, and thesubsequent release of N-acetyl glucosamine was determined using theassay described, above, in Section 18.1.

The results of these experiments indicated that partially deacetylatedmembranes are more susceptible to digestion by lysozyme (see FIG. 24)and, further, that the rate of lysozyme degradation is directly relatedto the extent of deacetylation (see FIG. 25, which compares thedegradation rates of a 50% to a 25% deacetylated p-GlcNAc membrane).

p-GlcNAc In Vivo Degradation

Additionally, experiments were performed which addressed the in-vivobiodegradability of p-GlcNAc materials. Such experiments utilized anabdominal implantation model. Three p-GlcNAc materials, as listed below,were tested.

p-GlcNAc Materials Tested:

-   -   1) p-GlcNAc, fully acetylated (designated prototype 1);    -   2) partially deacetylated p-GlcNAc membrane (designated        prototype 3A); and    -   3) lyophilized and partially deacetylated p-GlcNAc membrane        (designated prototype 4).

Results

The fully acetylated p-GlcNAc (prototype 1) was resorbed within 21 days,as shown in FIGS. 26A-26C. The partially deacetylated p-GlcNAc membrane(prototype 3A) was completely resorbed within 14 days, as shown in FIGS.26D-26E. The lyophilized and partially deacetylated p-GlcNAc membrane(prototype 4) had not yet been completely resorbed after 21 dayspost-implantation.

Histopathology analyses showed that once the p-GlcNAc material has beenresorbed there were no histological differences detectable betweentissue samples obtained from the treated and from the control animals.

19. EXAMPLE p-GlcNAc Hemostasis Studies

The experiments described herein study the efficacy of the p-GlcNAcmaterials of the invention for the control of bleeding. The success ofthe p-GlcNAc materials in controlling bleeding is, further, comparedagainst commercially available hemostatic products.

19.1. Materials and Methods

p-GlcNAc and control materials: partially deacetylated (approximately70%) p-GlCNAc membranes were made using the chemical separationtechnique described, above, in Section 5.3.2, with hydrofluoric acidbeing utilized as the chemical reagent, and the techniques described,above, in Section 5.4. 2 cm×1 cm pieces were used. p-GlcNAc-lactate gel(4% p-GlcNAc-lactate, formulated in propylene glycol and water) wasproduced following the methods described, above, in Section 17.1. Thecontrol material utilized for the study of bleeding in the spleen andliver was Gelfoam™ (Upjohn Company). Gelfoam™ and Avitene™ (MedchemProducts, Inc.) were the control materials used in the study of smallblood vessel bleeding.

Test animals: New Zealand White rabbits were used. 3 animals receivedtwo wounds in the spleen and one wound in the liver. 4 animals receivedfive surgical wounds to blood vessels of similar size in the caudalmesenteric artery system.

Surgical Preparation: The animals were anesthetized with ketamine HCland Xylazine. The animals were placed in dorsal recumbency, and all thehair from the abdomen was removed. The abdomen was then scrubbed withpovidone-iodine and 70% isopropyl alcohol and draped for asepticsurgery.

Liver/spleen surgical procedures: A midline incision was made and eitherthe spleen or liver was exteriorized and packed with moist sponges. A3-4 mm diameter cork borer was used to make a circular wound of about 2mm depth at one end of the organ. Once the splenic tissue was removed, apre-weighed 4″×4″ gauze sponge was used to absorb all the blood lostfrom the splenic wound for a period of one minute. The sponge wasre-weighed to quantify the amount of blood lost from that particularwound. The test animal was then treated by application to the wound ofone of the treatment materials. The time until hemostasis and the amountof blood lost prior to hemostasis was recorded.

After hemostasis in the first wound was achieved, a second wound in thespleen and one wound in the liver were made following the sameprocedure.

Small blood vessel surgical procedure: A midline incision was made andthe small bowel was exteriorized exposing the caudal mesenteric arterysystem. The bowel was packed with moist sponges and five blood vesselsof about the same size were identified. A scalpel was used to make awound of about 1 mm depth at one of the vessels. A pre-weighed 4″×4″gauze sponge was used to absorb all the blood lost from the vessel woundfor a period of one minute. The sponge was re-weighed to quantify theamount of blood lost from that particular wound. The animal was thentreated by application to the wound of one of the treatment materials.The time until hemostasis occurred and the amount of blood lost prior tohemostasis were recorded.

After hemostasis in the first wound was achieved, four more wounds weremade following the same procedure.

19.2. Results

p-GlcNAc materials were tested for their ability to control bleeding inthe spleen and liver of rat animal models. The p-GlcNAc materials testedwere: 1) partially deacetylated (approximately 70%) p-GlcNAc; and 2)p-GlcNAc-lactate gel (4% p-GlcNAc-lactate, formulated in propyleneglycol and water). The effectiveness of these p-GlcNAc materials wascompared to Gelfoam™ (Upjohn Company).

Each material was tested three times (twice in the spleen and once inthe liver). Both of the p-GlcNAq based materials exhibited aneffectiveness in controlling bleeding within the first minute afterapplication which was comparable to that of Gelfoam™. The p-GlcNAc basedmaterials have additional advantages. Specifically, the p-GlcNAcmaterials do not need to be held in place during the procedure, may beleft in the body, where they will be resorbed within two to three weeks(Gelfoam™ is not indicated for this purpose), are compatible with bothgeneral and minimally invasive surgical procedures.

Next, the efficacy of p-GlcNAc based materials in the control ofbleeding in small blood vessels was studied, and compared againstcommercially available hemostatic products.

Each material was tested five times (twice in one of the animals andonce in the other three animals). The p-GlcNAc membrane and gelformulations were easily applied to the site and controlled the bleedingwithin 2 minutes. Gelfoam™, which had to be held in place in order toperform its function achieved hemostasis within the same 2 minute rangeas the p-GlcNAc materials. Avitene™, a fibrous material made ofcollagen, was difficult to handle and required more than five minutes tocontrol the bleeding.

Thus, the results described herein demonstrate that the p-GlcNAcmaterials tested here represent effective, convenient hemostatic agents.

20. EXAMPLE p-GlcNAc Drug Delivery Systems

Described herein are studies demonstrating the successful use ofp-GlcNAc materials to deliver anti-tumor drugs to the site of malignantskin cancer and colon cancer tumors such that the delivered anti-tumordrugs exhibit a therapeutic impact upon the tumors.

20.1. Materials and Methods

p-GlcNAc-lactate drug delivery compositions: Mixtures of 5′-fluorouracil(5′-FU) and p-GlcNAc-lactate were formulated as follows; 0.5 mL of 5′-FU(50 mg/mL) was mixed with 0.5 mL of propylene glycol, and 2.0 mL of 4%p-GlcNAc-lactate was added and mixed. The p-GlcNAc-lactate was producedusing the techniques described, above, in Section 5.4. Even afterextensive mixing, the 5′FU did not completely dissolve into thep-GlcNAc-lactate gel. Assuming complete mixing, the final concentrationof 5′-FU would be 6.25 mg/mL.

Mixtures of mitomycin (Mito) and p-GlcNAc-lactate were formulated asfollows; 0.5 mg of Mito (lyophilized powder) were dissolved in 5 ml ofpropylene glycol, and 0.5 ml of the Mito solution was mixed with 0.5 mLof MPT's 4% p-GlcN-lactate preparation to give a final Mitoconcentration of 0.2 mg/ml and a final p-GlcNAc-lactate concentration of2%. The materials were compatible, with the Mito dissolving easily intothe p-GlcNAc-lactate gel.

p-GlcNAc membrane 5′FU delivery compositions: Samples of 5′-fluorouracil(5′-FU) were immobilized into disks of pure p-GlcNAc membrane materialproduced using the chemical separation method described, above, inSection 5.3.2, with hydrofluoric acid being utilized as the chemicalreagent. Each disk described here had a diameter of 1.5 cm, as describedhere.

For the preparation of high dose (HD) disks, 0.64 mL of a 50 mg/mLsolution of 5′-FU was mixed with suspensions containing approximately 8mg of pure p-GlcNAc. The mixtures were allowed to stand for severalhours to promote the absorption of 5′-FU into the p-GlcNAc, and werethen dried at 55° C. for 3.5 hours. The resulting HD disks contained atotal of 32 mg 5′-FU, which is equivalent to approximately twice thenormal total 14 day dose of 5′-FU typically given to a cancer patient,normalized to the weight of the experimental mice based on the typicaldose of 5′-FU per Kg body weight for humans. It should be noted here,and in the low dose concentrations described below, that the amount of5′-FU contained in the disks was approximated and was based only on theamount of 5′-FU put into the suspensions.

Low dose (LD) 5′-FU containing p-GlcNAc disks were prepared in the samemanner, except that the LD disks contained 17 mg of 5′-FU, an amountequivalent to equal the normal total human dose for a 14 day period,normalized to the weight of the experimental mice based on the typicaldose of 5′-FU per Kg body weight for humans.

Test Animals: For the 5′FU study, SCID (severe combinedimmunodeficiency) mice were inoculated with subcutaneous flankinjections of HT-29 colon cancer cells (ATCC; 1×10⁵ cells per inoculum)obtained by standard tissue culture methods, in order to produce HT-29colon cancer tumors. These injections led to palpable tumors which wereharvested in 14-21 days. Tumors were dissected and necrotic tissue wascut away. The HT-29 colon cancer tumors were sliced into 3×3×3 mmpieces.

The experimental SCID mice were anesthetized via intra-peritonealinjections with a standard dose of Avetin, and a slice of HT-29 coloncancer tumor was implanted onto the cecum of each mouse. Specifically,each mouse was surgically opened to expose its abdomen and the cecum waslocated, which was nicked with a scalpel to make a small incision. A3×3×3 mm tumor slice was sutured over the incision onto the cecum using5.0 silk sutures. The abdomen was then closed using Clay Adams staples.

All mice were caged singly and fed for two weeks. All mice were healthyand none had obstructed colons at the end of the two week period.

On day 14, each mouse was anesthetized, and its abdomen was reopened.The growing tumors were measured (length and horizontal dimensions).Tumors were then treated with the p-GlcNAc/anti-tumor drug or were usedas controls.

Six mice were used for the p-GlcNAc-lactate 5′FU study, and 15 mice wereused for the p-GlcNAc membrane 5′FU study.

For the mitomycin study, nine SCID mice were inoculated withsub-cutaneous injections of A431 squamous cell skin cancer cells (ATCC;1×10⁵ cells per inoculum). Tumors resulted in all mice within 14 days.

Treatments: For the p-GlcNAc-lactate 5′FU study, animals were treatedonce daily by “painting” the 5′-fluorouracil (5′-FU)-containing p-GlcNAcgel mixture onto the skin area over the tumor mass. Measurements of thetumor size were obtained daily. Control animals included animals treatedwith p-GlcNAc alone, without 5′-FU, and animals which received notreatment.

For the p-GlcNAc membrane ≡′FU study, the HT29 colon tumors in the SCIDmice were treated by surgically implanting disks of the drug-containingp-GlcNAc membrane material directly onto their surface, after havingallowed the tumor to grow on the colon for 14 days. Mice were sacrificed14 days following the implant procedure. Measurements of tumor volumeswere made immediately prior to implanting the drug-containing p-GlcNAcmembranes on day 0 and at the termination of the experiment on day 14.Control animals included ones treated with the p-GlcNAc membrane without5′-FU, and controls which received no treatment. Additionally, twoanimals received daily systemic injections of 5′-FU in doses equivalentto the HD and LD regimen.

For the p-GlcNAc-lactate Mito study, animals were treated daily as inthe p-GlcNAc-lactate 5′-FU study, with 3 animals being treated with theMito containing mixture. In addition, 3 animals were treated withp-GlcNAc minus Mito, 2 animals received no treatment, and 1 animalreceived propylene glycol.

20.2. Results 20.2.1. p-GlcNAc-Lactate 5′FU

Experiments designed to study the effect of p-GlcNAc-lactate Mito drugdelivery systems on tumor size were conducted, as described, above, inSection 20.1.

The largest length and width dimension were measured for each tumor andthe cross-sectional area using these dimensions was calculated. Thecross-sectional area values are shown in Table X, below. TABLE X Animal# Treatment Day 0 Day 4 Day 11 Day 15 Tumor Size (cm²) 1 CL + 5FU 63 90168 156 2 CL + 5FU 48 56 70 88 3 CL Control 21 36 88 108 4 CL Control 58110 150 195.30 5 Nothing 40 64 132 234 6 Nothing 28 42 100 132 %Increase in Size 1 CL + 5FU 0 43 167 147 2 CL + 5FU 0 17 47 84 3 CLControl 0 71 319 414 4 CL Control 0 90 160 289 5 Nothing 0 61 232 488 6Nothing 0 48 253 366

The data comparing p-GlcNAc-lactate 5′FU treated animals with controlsare shown in FIGS. 27-28. The data summarized in Table X and FIGS. 27-28clearly suggest that the HT-29 subcutaneous tumors in the rats treatedwith the 5′-FU containing p-GlcNAc-lactate gels have a significantlyretarded rate of growth compared to controls. Their growth has beenslowed 2.5-fold in comparison to the p-GlcNAc-lactate gel controls and4-fold compared to the no treatment controls.

20.2.2. p-GlcNAc-Lactate Mito

Experiments designed to study the effect of p-GlcNAc-lactate 5′FU drugdelivery systems on tumor size were also conducted, as described, above,in Section 20.1.

The largest length and width dimensions were measured for each tumor andthe cross sectional area using these dimensions was calculated. Thecross-sectional area values were as shown in Table XI, below. TABLE XITumor Size (cm²) Animal # Treatment Day 0 Day 3 Day 5 Day 8 1 pGlcNAc-23 23 42 49 Lactate + Mito 2 pGlcNAc- 23 16 54 63 Lactate + Mito 3pGlcNAc- 72 99 Term Term Lactate + Mito 4 pGlcNAc- 27 54 140 203 Lactatecontrol 5 pGlcNAc- 30 54 96 140 Lactate control 6 pGlcNAc- 30 58 200 221Lactate control 7 Nothing 48 75 126 300 8 Nothing 44 80 207 Dead 9Propylene 49 86 180 216 glycol

% Increase in Size Day 0 Day 3 Day 5 Day 8 1 pGlcNAc- 0 0 83 135Lactate + Mito 2 pGlcNAc- 0 −30 135 174 Lactate + Mito 3 pGlcNAc- 0 38Term Term Lactate + Mito 4 pGlcNAc- 0 100 419 652 Lactate control 5pGlcNAc- 0 80 220 367 Lactate control 6 pGlcNAc- 0 93 567 637 Lactatecontrol 7 Nothing 0 56 163 525 8 Nothing 0 82 370 Dead 9 Propylene 0 76267 341 glycol

The data comparing p-GlcNAc-lactate Mito treated animals with controlsare shown in FIGS. 29-30. The data summarized in Table XI and FIGS.29-30 clearly suggest that the tumors growing in the rats treated withthe Mitomycin-containing p-GlcNAc-lactate gels animals have asignificantly retarded rate of growth. Their growth was slowed 4-fold incomparison to the p-GlcNAc-lactate gel controls and 4-fold compared tothe no treatment controls.

20.2.3. p-GlcNAc Membrane 5′FU

Next, experiments designed to study the effect of p-GlcNAc membrane 5′FUdrug delivery systems on skin cancer tumor size were conducted, asdescribed, above, in Section 20.1.

The tumor volume data obtained during the study, including percentchange in volume caused by the different treatments, is summarized inTable XII, below. Tumors were assumed to be cylindrical in shape. Theirvolumes were determined by measuring their width and length, and usingthe following equation: V=πr²l, where the radius r is 0.5 times thewidth and l is the length. TABLE XII p-GlcNAc Membrane + 5′-FluorouracilAnimal Data Tumor Volume Tumor Volume Pre-Treatment Post-treatmentAnimal # Treatment (mm³) (mm³) % Change Comments A. 5′-FU High Dose: 1.5′-FU HD 393 283 −28.0 2. 5′-FU HD 785 308 −60.8 3. 5′-FU HD 98.1 62.8−36.0 4. 5′-FU HD 785 550 −30.0 Average per Animal −38.7 B. 5′-FU LowDose: 5. 5′-FU LD 603 170 −71.9 6. 5′-FU LD 603 615 2.0 7. 5′-FU LD 269198 −26.0 8. 5′-FU LD 169 226 33.3 Average per animal −15.7 C. p-GlcNAcControl: 9. p-GLcNAc Control 170 550 320.0 10. p-GLcNAc Control Died day12 Average per Animal 320.0 D. No Treatment Control: 11. No Treatment402 864 215.0 Sat. tumors 12. No Treatment 21.2 572 2700.0 Sat. tumorsAverage per Animal 1457.0 E. 5′-FU via Intravenous Injection - Control:13. Low Dose 402 703 175.0 14. Low Dose 402 402 0.0 Died day 13 15. HighDose 402 132 −67.0 Died day 13

FIG. 31 summarizes a portion of the data presented, above, in Table XII.as shown in FIG. 31, the data strongly suggest that tumors treated withthe high dose (HD) 5′-FU-containing p-GlcNAc membranes have stoppedgrowing and have, in all cases, actually become significantly smaller.The low dose (LD) polymer materials resulted in disease stability andslight decrease in tumor size. In contrast, the tumors in the controlanimals continued to rapidly increase in size. It is interesting to notethat two of the three control animals which were treated via IV diedduring the study, indicating that systemic delivery of the equivalentamount of 5′-FU is lethal, whereas site-specific delivery via thep-GlcNAc polymer is efficacious in ridding the animal of the disease.

20.3. Conclusion

The data presented in this Section strongly suggest that thesite-specific delivery of anti-tumor drugs has a positive effect inretarding and reversing tumor growth. Successful results were obtainedusing p-GlcNAc drug delivery compositions having two differentformulations, namely p-GlcNAc-lactate and p-GlcNAc membraneformulations. Further, successful results were obtained using twodifferent anti-tumor drugs, 5′-FU and Mito. Thus, the p-GlcNAc drugdelivery systems of the invention exhibit anti-tumor activity, useful,for example, in the delivery of drugs specifically to the site of thetumor cells of interest.

21. EXAMPLE Further p-GlcNAc Drug Delivery Systems

Described herein are further studies demonstrating the successful use ofp-GlcNAc materials to deliver 5′-FU to the site of malignant coloncancer tumors such that the delivered 5′-FU exhibit a therapeutic impactupon the tumors.

21.1. Materials and Methods

p-GlcNAc membrane 5′FU delivery composition:

The p-GlcNAc membrane 5′-FU delivery composition was formulated asdescribed herein.

The aqueous p-GlcNAc suspension used was a 1.0-1.1 mg/ml mixture. 0.70ml of the p-GlcNAc suspension was filtered using a glass frittered diskfunnel and a 0.8 micron, 47 mm membrane filter. After approximately 5-6minutes on the filtering apparatus, the membrane filter, containing thep-GlcNAc, was removed and placed in a clean petri dish. Using a cleanscalpel and forceps, the p-GlcNAc was scraped gently from the membraneand placed into a 2 ml cryovial. At this time, the appropriate amount of5′-FU (i.e., 50 mg/ml) was added to the p-GlcNAc in the cryovial. Forpreparation of the control disk, no 5′-FU was added.

For example, to prepare a 0.5× disk, 0.17 ml of 5′-FU (50 mg/ml 5′-FUsolution) was added for a total of 8 mg of 5′-FU/disk. The cryovial wasthen securely capped and the contents vortexed until a thick slurryformed. The slurry was then placed into one well of a 48-well plate, andflattened using a glass stirring rod. The above procedure was thenrepeated until the desired number of p-GlcNAc disks were prepared. Afterall the disks were placed in the 48-well plate, the plate was coveredwith Parafilm™ and the contents were frozen for at least one hour. Then,the disks were lyophilized overnight (approximately sixteen hours).Using a sterile needle, the disks were freed from the 48-well plate andflattened using the bottom of a clean dish and light pressure by hand.The disks were stored in clean petri dishes and labeled. It should benoted that autoclaving (e.g., 20 min, 121° C.) the disks does not damagethem.

The 1× disks were prepared in the same manner as above except that 0.34ml of 5′-FU (50 mg/ml 5′-FU solution) was added for a total of 16 mg of5′-FU/disk. To prepare the 2× disks, 0.68 ml of 5′-FU (50 mg/ml of 5′-FUsolution) was added for a total of 32 mg of 5′-FU/disk.

The resulting 0.5× disk contained a total of 8 mg 5′-FU each, which isequivalent to approximately half the normal total 14 day dosage of 5′-FUtypically given to a cancer patient by intravenous administration,normalized to the weight of the experimental mice based on the typicaldose of 5′-FU per kg of body weight for humans. 1× p-GlcNAc diskscontaining 5′-FU were prepared in the same manner, except that the 1×p-GlcNAc disks contained 16 mg of 5′-FU, an amount equivalent to equalthe normal total 14 day dose of 5′-FU typically given to a cancerpatient by intravenous administration, normalized to the weight of theexperimental mice based on the typical dose of 5′-FU per kg body weightfor humans. 2× p-GlcNAc disks contained 32 mg of 5′-FU, an amount equalto twice times the normal total 14 day dose of 5′-FU typically given toa cancer patient by intravenous administration, normalized to the weightof the experimental mice based on the typical dose of 5′-FU per kg bodyweight for humans.

Test Animals

The test animals for the present 5′-FU study on HT-29 colon tumors wereprepared in the same manner as described in Section 20.1.

Treatments

The mice were treated in the same manner as described in Section 20.1for the p-GlcNAc membrane 5′-FU study, except that the mice weresacrificed 10 days following the implant procedure.

21.2. Results

The tumor volume data obtained during the study included percent changein volume caused by the different treatments, is summarized below inTable XIII. Tumors were assumed to be cylindrical in shape. Theirvolumes were determined by measuring their width and length, and usingthe equation: V=πr²l, where the radius, r, is 0.5 times the width and lis the length. TABLE XIII Tumor Volume Pre- Tumor Volume Animal #Treatment Treatment (mm³) Post-treatment (mm³) % Change Comments A. 0.5xDose: 1. 5′-FU (.5x) 169.0 — — Died post-op 2. 5′-FU (.5x) 785.0 401.9−48 3. 5′-FU (.5x) 863.0 141.3 −83 Average per Animal −65.5 B. 1x Dose:4. 5′-FU (1.0x) 269.0 98 −63 Died Day 8 post membrane 5. 5′-FU (1.0x)269.0 — — Died Post-Op 6. 5′-FU (1.0x) 401.9 62 −84 Died Day 8 postmembrane Average per Animal −73.5 C. 2x Dose: 7. 5′-FU (2.0x) 401.9 98.0−75 Died Day 8 post membrane 8. 5′-FU (2.0x) 785 50.24 −93 Died Day 8post membrane 9. 5′-FU (2.0x) 572 — — Died post-op Average per Animal−84.0 D. p-GlcNAc Control: 10. p-GlcNAc Control 401.9 785.0 +190 11.p-GlcNAc Control 1356.5 1140.0 +30 2nd mass 385.0 Average per Animal+110 E. No Treatment Control: 12. No Treatment 401.9 1020.5 +250 13. NoTreatment 50.0 572.0 +1100 Average per Animal +675

21.3. Conclusion

As is shown in Section 21.2, above, each of the disks (i.e., 1× and 2×)were very effective in reducing tumor size. While the 0.5× dose disksdid so with little noticeable toxicity, the 1× and 2× dose disksresulted in significant mortality rates. This is presumably due to thehigher effective 5′-FU dose being delivered to the animals. Thus,according to this data, it appears that doses smaller than conventionaldoses (in this case half the conventional dose) provide site-deliveryeffective in reducing tumor size without toxic effects.

Furthermore, preparation of the disks as described in this Example, incomparison to the Example presented in Section 20, below, appears toproduce disks with less variability in dosage. This observation is basedon the lack of toxicity found in the data presented in Section 20(suggesting less incorporation of 5′-FU then originally presumed) andthe variability in tumor reduction. For example, in Section 20, TableXII shows a wide range of tumor size changes, while in contrast, no suchlarge variations were noted in this Section, as exemplified in TableXIII, above.

22. EXAMPLE Wound Healing Studies

The experiment described herein studies the efficacy of the p-GlcNAcmaterials of the invention for the promotion of wound healing and thereduction of scar tissue.

22.1. Materials and Methods

p-GlcNAc membrane: p-GlcNAc membranes (2.0×2.0 cm) were used in thisstudy. The p-GlcNAc was prepared using the chemical purification methoddescribed, above, in Section 5.3.2.

Test Animals: Four healthy 23-27 kg, 13 week old Yorkshire pigs wereused in the study. The animals were held in quarantine for seven daysprior to the surgical procedure. Each animal was identified with anindividual permanent ear tag.

Experimental Design: Four healthy Yorkshire pigs were used with eachanimal serving as its own control. Under general anesthesia four 2.0×2.0cm serosal abrasion lesions were created on the surface of the intestinein each animal. Lesions 1 and 3 were left as untreated control sites,while sites 2 and 4 were each topically treated with the test article.Animals were recovered for 21 days at which time they werereanesthetized. The abdomen was explored and the abrasion sites wereexamined macroscopically for hemorrhaging, necrosis, discolorations andfibroblasts. Tissue samples were collected for histomicroscopy andscanning electron microscopy. The animals were anesthetized using acombination of xylazine (3.5-5.5 mg/kg, body weight) and Telazol(5.5-8.0 mg/kg, body weight) administered intra-muscularly. Animals werethen incubated and maintained under general anesthesia using halothaneand oxygen.

Surgical Procedure: With the animal under general anesthesia and indrosal recumbency, the ventral abdomen was aseptically prepared forsurgery. The abdomen was clipped and then a gross and sterile scrub wasperformed using povidone iodine and 70% isopropyl alcohol. The animalwas then draped for surgery. A 12 cm ventral midline celiotomy was made.Four (2.0×2.0 cm) serosal abrasions were created by lightly passing anew scalpel blade (#10 Bard-Parker blade, using 1 blade/site) across theserosal surface for 200 counts. Lesions created consisted of uniformlydistributed minute, petechial hemorrhages. The first two lesions werecreated on the antimesenteric serosal surface of the small intestine andthe remaining two were created on the antimesenteric serosal surface ofthe spiral colon. The margins of each lesion site were identified byusing 4 peripherally placed sutures of 3-0 nylon. Sites 1 and 3 wereleft untreated and sites 2 and 4 were treated with the test article. Theabdomen was closed using PDS and stainless steel staples for skinclosure.

Clinical Observations were performed and recorded daily.

Euthanasia was achieved by administering an overdose of barbituratewhile the animal was under general anesthesia on Day 21. The procedurewas performed following the American Veterinary Medical Associationguidelines.

22.2. Results

The treatment with the p-GlcNAc membrane was beneficial in promotingwound healing and reducing scar tissue formation. Macroscopically, thenon-treated control sites were characterized by extensive fibrosis anddiscoloration. In contrast, the p-GlcNAc treated sites developed healthytissue and very limited fibrosis. Representative findings areillustrated in FIGS. 32A and 32B. (The spots are the remains of thesuture material that was used to identify the margins of the lesions.)Histomicroscopy and scanning electron microscopy findings wereconsistent with the macroscopic observations.

23. EXAMPLE Taxol Formulation

Presented in this Example is a method of preparing variousconcentrations of taxol-pGlcNAc formulations.

23.1. Materials and Methods

R-GlcNAc starting material: An aqueous p-GlcNAc suspension (1.0-1.1mg/ml mixture) was used. 0.70 ml of the aqueous p-GlcNAc solution wasfiltered using a glass filtered disk funnel and a 0.8 micron, 47 mmmembrane filter. After approximately 5-6 minutes on the filteringapparatus, the membrane filter, containing the p-GlcNAc, was removed andplaced in a clean petri dish. Using a clean scalpel and forceps, thep-GlcNAc was scraped gently from the membrane and placed into a 2 mlcryovial. At this time, the appropriate amount of taxol (6 mg/ml) andH₂O (deionized and distilled) was added to the p-GlcNAc in the cryovial.

For example, as shown in Table XIV, below, to prepare a 1× diskcontaining 0.21 mg of taxol, 35 μl of taxol (6 mg/ml taxol solution) and105 μl of H₂O were added. The cryovial was then securely capped and thecontents vortexed until a thick slurry formed. The slurry was thenplaced into a well of a 48-well plate, and flattened using a glassstirring rod. The above procedure was then repeated until the desirednumber of p-GlcNAc disks were prepared.

After all the disks were placed in the 48-well plate, the plate wascovered with Parafilm™ and the contents were frozen for at least onehour. Then, the disks were lyophilized overnight (approximately sixteenhours). Using a sterile needle, the disks were freed from the 48-wellplate and flattened using the bottom of a clean dish and light pressureby hand. Disks were stored in clean petri dishes and labeled. It shouldbe noted that autoclaving (e.g., 20 min, 121° C.) the disks does notharm them.

Similarly, a 2× formulation containing 0.42 mg of taxol, was prepared asabove, except that 701 of taxol (mg/ml taxol solution) and 105 μl H₂Owas added to the p-GlcNAc in the cryovial. Table XIV, below, summarizesvarious taxol/p-GlcNAc formulations. TABLE XIV Dose of Taxol p-GlcNAcH₂O added Controls - No Taxol 7 ml p-GlcNAc 140 μl H₂O 1X - 35 μl (0.21mg) Taxol 7 ml p-GlcNAc 105 μl H₂O 2X - 70 μl (0.42 mg)Taxol 7 mlp-GlcNAc  70 μl H₂O 4X - 140 μl (0.84 mg)Taxol 7 ml p-GlcNAc  0 μl H₂O

It is apparent that many modifications and variations of this inventionas set forth here may be made without departing from the spirit andscope thereof. The specific embodiments described above are given by wayof example only, and the invention is limited only by the terms of theappended claims.

1. A biocompatible and immunoneutral poly-β-1→4-N-acetylglucosamine. 2.A biocompatible and immunoneutral poly-β-1→4-glucosamine.
 3. Abiocompatible poly-β-1→4-N-acetylglucosamine whose polysaccharide chainis hydrolyzed.
 4. A biocompatible poly-β-1→4-glucosamine whosepolysaccharide chain is hydrolyzed.
 5. Thepoly-β-1→4-N-acetylglucosamine of claim 1 or 3, wherein at least one ofthe N-acetylglucosamine monosaccharides has been deacetylated.
 6. Thepoly-β-1→4-N-acetylglucosamine of claim 5, wherein at least about 25% toabout 75% of the N-acetylglucosamine monosaccharides have beendeacetylated.
 7. The biocompatible poly-β-1→4-N-acetylglucosamine ofclaim 3 whose polysaccharide chain is partially hydrolyzed.
 8. Thebiocompatible poly-β-1→4-glucosamine of claim 4 whose polysaccharidechain is partially hydrolyzed.
 9. The biocompatiblepoly-β-1→4-N-acetylglucosamine of claim 3 whose polysaccharide chain iscompletely hydrolyzed.
 10. The biocompatible poly-β-1→4-glucosamine ofclaim 4 whose polysaccharide chain is completely hydrolyzed.